Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acids

ABSTRACT

The present invention is directed to nucleic acid molecules and nucleic acid constructs, and other agents associated with fatty acid synthesis, particularly the ratios of saturated and unsaturated fats. Moreover, the present invention is directed to plants incorporating such agents where the plants exhibit altered ratios of saturated and unsaturated fats. In particular, the present invention is directed to plants incorporating such agents where the plants exhibit altered ratios of monounsaturated to polyunsaturated fatty acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This national stage application is a continuation of InternationalApplication PCT/US03/19445, filed Jun. 20, 2003, which is acontinuation-in-part of U.S. patent application Ser. No. 10/176,149,filed Jun. 21, 2002, now U.S. Pat. No. 7,067,722, which is acontinuation-in-part of U.S. patent application Ser. No. 09/638,508,filed Aug. 11, 2000, now abandoned, which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Application Ser. No. 60/172,128,filed Dec. 17, 1999, and U.S. Provisional Application Ser. No.60/151,224, filed Aug. 26, 1999.

FIELD OF THE INVENTION

The present invention is directed to nucleic acid molecules and nucleicacid constructs, and other agents associated with fatty acid synthesis.Moreover, the present invention is directed to plants incorporating suchagents where the plants exhibit altered ratios of saturated andunsaturated fats. In particular, the present invention is directed toplants incorporating such agents where the plants exhibit altered ratiosof monounsaturated to polyunsaturated fatty acids.

BACKGROUND

Plant oils are used in a variety of applications. Novel vegetable oilcompositions and improved means to obtain oil compositions, frombiosynthetic or natural plant sources, are needed. Depending upon theintended oil use, various different fatty acid compositions are desired.

Higher plants appear to synthesize fatty acids via a common metabolicpathway—the fatty acid synthetase (FAS) pathway. In developing seeds,where fatty acids are attached to glycerol backbones, formingtriacylglycerides, for storage as a source of energy for furthergermination, the FAS pathway is located in the plastids. The firstcommitted step is the formation of acetyl-ACP (acyl carrier protein)from acetyl-CoA and ACP catalyzed by the enzyme, acetyl-CoA:ACPtransacylase (ATA). Elongation of acetyl-ACP to 16- and 18-carbon fattyacids involves the cyclical action of the following sequence ofreactions: condensation with a two-carbon unit from malonyl-ACP to forma β-ketoacyl-ACP (β-ketoacyl-ACP synthase), reduction of theketo-function to an alcohol (β-ketoacyl-ACP reductase), dehydration toform an enoyl-ACP (β-ketoacyl-ACP dehydratase), and finally reduction ofthe enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACPreductase). β-ketoacyl-ACP synthase I catalyzes elongation from C4:0 upto palmitoyl-ACP (C16:0), whereas β-ketoacyl-ACP synthase II catalyzesthe final elongation to stearoyl-ACP (C18:0). Common plant unsaturatedfatty acids, such as oleic, linoleic and linolenic acids found instorage triacylglycerides, originate from the desaturation ofstearoyl-ACP to form oleoyl-ACP (C18:1) in a reaction catalyzed by asoluble plastid Δ-9 desaturase (also often referred to as “stearoyl-ACPdesaturase”). Molecular oxygen is required for desaturation in whichreduced ferredoxin serves as an electron co-donor. Additionaldesaturation is effected sequentially by the actions of membrane boundΔ-12 desaturase and Δ-15 desaturase. These “desaturases” thus createpolyunsaturated fatty acids.

Obtaining nucleic acid sequences capable of producing a phenotypicresult in FAS, desaturation and/or incorporation of fatty acids into aglycerol backbone to produce an oil is subject to various obstaclesincluding but not limited to the identification of metabolic factors ofinterest, choice and characterization of an enzyme source with usefulkinetic properties, purification of the protein of interest to a levelwhich will allow for its amino acid sequencing, utilizing amino acidsequence data to obtain a nucleic acid sequence capable to use as aprobe to retrieve the desired DNA sequence, and the preparation ofconstructs, transformation and analysis of the resulting plants.

Thus, additional nucleic acid targets and methods for modifying fattyacid compositions are needed. In particular, constructs and methods toproduce a range of different fatty acid compositions are needed.

SUMMARY OF THE INVENTION

The present invention provides a substantially purified nucleic acidmolecule comprising a nucleic acid sequence with at least 70% sequenceidentity to a nucleic acid sequence selected from the group consistingof SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 4, complementsthereof, and fragments of either.

Further provided by the present invention are a nucleic acid moleculecomprising at least 15 consecutive nucleotides of a nucleic acidmolecule having the sequence of SEQ ID NO:12; a nucleic acid moleculecomprising at least 15 consecutive nucleotides of a nucleic acidmolecule having the sequence of SEQ ID NO:13; a nucleic acid moleculecomprising at least 15 consecutive nucleotides of a nucleic acidmolecule having the sequence of SEQ ID NO:14; and a nucleic acidmolecule comprising at least 15 consecutive nucleotides of a nucleicacid molecule having the sequence of SEQ ID NO:4.

Also provided by the present invention is a recombinant nucleic acidmolecule comprising as operably linked components: (A) a promoter thatfunctions in a plant cell to cause production of an mRNA molecule; and(B) a nucleic acid sequence that hybridizes under high stringencyconditions to a nucleic acid sequence selected from the group consistingof SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 4, complementsthereof, and fragments of either.

Further provided by the present invention is a transformed soybean planthaving a nucleic acid molecule that comprises (a) a first promoteroperably linked to a first nucleic acid molecule having a first nucleicacid sequence that has 85% or greater identity to a nucleic acidsequence selected from the group consisting of SEQ ID NO:1 through SEQID NO:2, complements thereof, and fragments of either, and (b) a secondnucleic acid molecule having a second nucleic acid sequence that has 85%or greater identity to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:4 through SEQ ID NO:14, complements thereof,and fragments of either, wherein the second nucleic acid molecule isoperably linked to the first promoter in a polycistronic configurationor to a second promoter.

Further provided by the present invention is a transformed soybean plantcomprising a double-strand RNAi construct where a first promoter isoperably linked to a first nucleic acid molecule, having a first nucleicacid sequence that has 85% or greater identity to a nucleic acidsequence selected from the group consisting of SEQ ID NO:1 through SEQID NO:2, complements thereof, and fragments of either, wherein a secondnucleic acid molecule having a second nucleic acid sequence that has 85%or greater identity to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:4 through SEQ ID NO:14, complements thereof, andfragments of either, is operably linked to the first nucleic acidmolecule.

Also provided by the present invention is a transformed soybean plantcomprising a double-strand RNAi construct where a first promoter isoperably linked to a first nucleic acid molecule, having a first nucleicacid sequence that has 85% or greater identity to a nucleic acidsequence selected from the group consisting of SEQ ID NO:1 through SEQID NO:2, complements thereof, and fragments of either, wherein a secondnucleic acid molecule having a second nucleic acid sequence that has 85%or greater identity to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:4 through SEQ ID NO:14, complements thereof, andfragments of either is operably linked to a second promoter in a dsRNAiconfiguration.

Also provided by the present invention is a transformed soybean planthaving two or more nucleic acid molecules wherein each nucleic acidmolecule is operably linked to a promoter and wherein each nucleic acidmolecule has a nucleic acid sequence that has 85% or greater identity toa nucleic acid sequence selected from the group consisting of SEQ IDNOs:1, 2, 4-14, complements thereof, and fragments of either.

The present invention provides a transformed soybean plant, wherein thelevel of a transcript encoded by a gene selected from the groupconsisting of FAD2-1A, FAD2-1B, FAD2-2B, FAD3-1A, FAD3-1B, FAD3-1C isselectively reduced while leaving the level of a transcript encoded by adifferent gene selected from the group consisting of FAD2-1A, FAD2-1B,FAD2-2B, FAD3-1A, FAD3-1B, FAD3-1C at least partially unaffected.

The present invention also provides a method of producing a soybeanplant having a seed with reduced linolenic acid content comprising:transforming a soybean plant with a nucleic acid molecule that comprises(a) a first promoter operably linked to a first nucleic acid moleculehaving a first nucleic acid sequence that has 85% or greater identity toa nucleic acid sequence selected from the group consisting of SEQ IDNO:1 through SEQ ID NO:2, complements thereof, and fragments of either,and (b) a second nucleic acid molecule having a second nucleic acidsequence that has 85% or greater identity to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:4 through SEQ ID NO:14,complements thereof, and fragments of either, wherein the second nucleicacid molecule is operably linked to the first promoter or a secondpromoter; and growing said plant, wherein said plant produces seed withless linolenic acid than a plant having a similar genetic background butlacking said nucleic acid molecule.

The present invention also provides a method of producing a soybeanplant having a seed with increased oleic acid content comprising:transforming a soybean plant with a nucleic acid molecule that comprises(a) a promoter operably linked to a first nucleic acid molecule having afirst nucleic acid sequence that has 85% or greater identity to anucleic acid sequence selected from the group consisting of SEQ ID NO:1through SEQ ID NO:2, complements thereof, acid fragments of either, and(b) a second nucleic acid molecule having a second nucleic acid sequencethat has 85% or greater identity to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:4 through SEQ ID NO:14,complements thereof, and fragments of either, wherein the second nucleicacid molecule is operably linked to the first promoter or a secondpromoter; and growing said plant, wherein said plant produces seed withmore oleic acid than a plant having a similar genetic background butlacking said nucleic acid molecule.

The present invention also provides a method of producing a plant havinga seed with a modified oil composition comprising: transforming a plantwith a nucleic acid molecule that comprises, as operably linkedcomponents, a first promoter and a first nucleic acid molecule having afirst nucleic acid sequence that has 85% or greater identity to anucleic acid sequence selected from the group consisting of SEQ ID NO:1, 2, 4 through 14, complements thereof, and fragments of either; and,growing said plant, wherein said plant produces seed with a modified oilcomposition compared to a plant having a similar genetic background butlacking said nucleic acid molecule.

The present invention further provides a method of producing a planthaving a seed with an altered ratio of monounsaturated topolyunsaturated fatty acids comprising: transforming a plant with aconstruct that comprises, as operably linked components, two or morenucleic acid molecules, each having a nucleic acid sequence that has 85%or greater identity to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, 2, 4 through 14, complements thereof, andfragments of either, wherein each nucleic acid molecule is operablylinked to a promoter; and, growing said plant, wherein said plantproduces seed with an altered ratio of monounsaturated topolyunsaturated fatty acids compared to a plant having a similar geneticbackground but lacking said two or more nucleic acid molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of construct pCGN5468.

FIG. 2 is a schematic of construct pCGN5469.

FIG. 3 is a schematic of construct pCGN5471.

FIG. 4 is a schematic of construct pCGN5485.

FIG. 5 is a schematic of construct pCGN5486.

FIG. 6 is a schematic of construct pCGN5462.

FIG. 7 is a schematic of construct pCGN5466.

FIG. 8 is a schematic of construct pCGN5464.

FIG. 9 is a schematic of construct pCGN5473.

FIG. 10 is a schematic of construct pMON68521.

FIG. 11 is a schematic of construct pMON68519.

FIG. 12 is a schematic of construct pCGN5455.

FIG. 13 is a schematic of construct pCGN5459.

DETAILED DESCRIPTION OF THE INVENTION

Description of the Nucleic Acid Sequences

SEQ ID NO:1 sets forth a nucleic acid sequence of a FAD2-1A intron 1.

SEQ ID NO:2 sets forth a nucleic acid sequence of a FAD2-1B intron 1.

SEQ ID NO:3 sets forth a nucleic acid sequence of a partial FAD2-2genomic clone.

SEQ ID NO:4 sets forth a nucleic acid sequence of a FAD2-2B intron 1.

SEQ ID NO:5 sets forth a nucleic acid sequence of a FAD3-1A intron 1.

SEQ ID NO:6 sets forth a nucleic acid sequence of a FAD3-1A intron 2.

SEQ ID NO:7 sets forth a nucleic acid sequence of a FAD3-1A intron 3A.

SEQ ID NO:8 sets forth a nucleic acid sequence of a FAD3-1A intron 4.

SEQ ID NO:9 sets forth a nucleic acid sequence of a FAD3-1A intron 5.

SEQ ID NO:10 sets forth a nucleic acid sequence of a FAD3-1A intron 3B.

SEQ ID NO:11 sets forth a nucleic acid sequence of a FAD3-1A intron 3C.

SEQ ID NO:12 sets forth a nucleic acid sequence of a FAD3-1B intron 3C.

SEQ ID NO:13 sets forth a nucleic acid sequence of a FAD3-1B intron 4.

SEQ ID NO:14 sets forth a nucleic acid sequence of a FAD3-1C intron 4.

SEQ ID NO:15 sets forth a cDNA sequence of a FAD2-1A gene sequence.

SEQ ID NOs:16 and 17 set forth nucleic acid sequences of FAD2-1A PCRprimers.

SEQ ID NO:18 sets forth a nucleic acid sequence of a partial FAD2-1Agenomic clone.

SEQ ID NO:19 sets forth a nucleic acid sequence of a partial FAD2-1Bgenomic clone.

SEQ ID NOs:20 and 21 set forth nucleic acid sequences of FAD3-1A PCRprimers.

SEQ ID NO:22 sets forth a nucleic acid sequence of a FAD2-1B promoter.

SEQ ID NO:23 sets forth a nucleic acid sequence of a partial FAD3-1Agenomic clone.

SEQ ID NOs:24 through 39 set forth nucleic acid sequences of PCRprimers.

Definitions

As used herein, the term “gene” is used to refer to the nucleic acidsequence that encompasses the 5′ untranslated region, including promoterregion, associated with the expression of the gene product, any intronand exon regions and 5′ or 3′ untranslated regions associated with theexpression of the gene product.

As used herein, a “FAD2”, “Δ12 desaturase” or “omega-6 desaturase” geneis a gene that encodes an enzyme capable of catalyzing the insertion ofa double bond into a fatty acyl moiety at the twelfth position countedfrom the carboxyl terminus.

When referring to proteins and nucleic acids herein, the use of plaincapitals, e.g., “FAD2”, indicates a reference to an enzyme, protein,polypeptide, or peptide, and the use of italicized capitals, e.g.,“FAD2”, indicates a reference to nucleic acids, including withoutlimitation genes, cDNAs, and mRNAs.

As used herein the terminology “FAD2-1” is used to refer to a FAD2 genethat is naturally expressed in a specific manner in seed tissue.

As used herein the terminology “FAD2-2” is used to refer a FAD2 genethat is (a) a different gene from a FAD2-1 gene and (b) is naturallyexpressed in multiple tissues, including the seed.

As used herein, a “FAD3”, “Δ15 desaturase” or “omega-3 desaturase” geneis a gene that encodes an enzyme capable of catalyzing the insertion ofa double bond into a fatty acyl moiety at the fifteenth position countedfrom the carboxyl terminus.

As used herein the terminology “FAD3-1” is used to refer a FAD3 genethat is naturally expressed in multiple tissues, including the seed.

As used herein the capital letter that follows the gene terminology (A,B, C) is used to designate the family member, i.e. FAD2-1A is adifferent gene family member from FAD2-1B.

As used herein, a “mid-oleic soybean seed” is a seed having between 50%and 75% oleic acid present in the oil composition of the seed.

As used herein, a “high oleic soybean seed” is a seed with oil havinggreater than 75% oleic acid present in the oil composition of the seed.

The term “non-coding” refers to sequences of nucleic acid molecules thatdo not encode part or all of an expressed protein. Non-coding sequencesinclude but are not limited to introns, promoter regions, 3′untranslated regions, and 5′ untranslated regions.

The term “intron” as used herein refers to the normal sense of the termas meaning a segment of nucleic acid molecules, usually DNA, that doesnot encode part of or all of an expressed protein, and which, inendogenous conditions, is transcribed into RNA molecules, but which isspliced out of the endogenous RNA before the RNA is translated into aprotein.

The term “exon” as used herein refers to the normal sense of the term asmeaning a segment of nucleic acid molecules, usually DNA, that encodespart of or all of an expressed protein.

As used herein, a promoter that is “operably linked” to one or morenucleic acid sequences is capable of driving expression of one or morenucleic acid sequences, including multiple coding or non-coding nucleicacid sequences arranged in a polycistronic configuration.

A “polycistronic gene” or “polycistronic mRNA” is any gene or mRNA thatcontains transcribed nucleic acid sequences which correspond to nucleicacid sequences of more than one gene targeted for expression. It isunderstood that such polycistronic genes or mRNAs may contain sequencesthat correspond to introns, 5′UTRs, 3′UTRs, or combinations thereof, andthat a recombinant polycistronic gene or mRNA might, for example withoutlimitation, contain sequences that correspond to one or more UTRs fromone gene and one or more introns from a second gene.

As used herein, the term complement of a nucleic acid sequence refers tothe complement of the sequence along its complete length.

As used herein, any range set forth is inclusive of the end points ofthe range unless otherwise stated.

Agents

The agents of the invention will preferably be “biologically active”with respect to either a structural attribute, such as the capacity of anucleic acid molecule to hybridize to another nucleic acid molecule, orthe ability of a protein to be bound by an antibody (or to compete withanother molecule for such binding). Alternatively, such an attribute maybe catalytic and thus involve the capacity of the agent to mediate achemical reaction or response. The agents will preferably be“substantially purified.” The term “substantially purified,” as usedherein, refers to a molecule separated from substantially all othermolecules normally associated with it in its native environmentalconditions. More preferably a substantially purified molecule is thepredominant species present in a preparation. A substantially purifiedmolecule may be greater than 60% free, greater than 75% free, preferablygreater than 90% free, and most preferably greater than 95% free fromthe other molecules (exclusive of solvent) present in the naturalmixture. The term “substantially purified” is not intended to encompassmolecules present in their native envirorunental conditions.

The agents of the invention may also be recombinant. As used herein, theterm “recombinant” means any agent (e.g., including but limited to DNA,peptide), that is, or results, however indirectly, from humanmanipulation of a nucleic acid molecule.

It is understood that the agents of the invention may be labeled withreagents that facilitate detection of the agent (e.g., fluorescentlabels, Prober et al., Science 238:336-340 (1987); Albarella et al., EP144914; chemical labels, Sheldon et al., U.S. Pat. No. 4,582,789;Albarella et al., U.S. Pat. No. 4,563,417; modified bases, Miyoshi etal., EP 119448).

Nucleic Acid Molecules

Agents of the invention include nucleic acid molecules. In an aspect ofthe present invention, the nucleic acid molecule comprises a nucleicacid sequence, which when introduced into a cell or organism, is capableof selectively reducing the level of a protein and/or transcript encodedby a FAD2 or FAD3 gene while leaving the level of a protein and/ortranscript encoded by a second FAD2 or FAD3 gene partially unaffected.In a preferred aspect of the present invention, the nucleic acidmolecule comprises a nucleic acid sequence, which when introduced into acell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD2 or FAD3 gene while leavingthe level of a protein and/or transcript encoded by a second FAD2 orFAD3 gene substantially unaffected. In a highly preferred aspect of thepresent invention, the nucleic acid molecule comprises a nucleic acidsequence, which when introduced into a cell or organism, is capable ofselectively reducing the level of a protein and/or transcript encoded bya FAD2 or FAD3 gene while leaving the level of a protein and/ortranscript encoded by a second FAD2 or FAD3 gene essentially unaffected.

In a preferred aspect, the capability of a nucleic acid molecule toselectively reduce the level of a gene relative to another gene iscarried out by a comparison of levels of mRNA transcripts. In anotherpreferred aspect of the present invention, the nucleic acid molecule ofthe invention comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:1 through 15, 18, 19, 22, 23, complementsthereof, and fragments of either. In another preferred aspect of thepresent invention the nucleic acid molecule of the invention comprises anucleic acid sequence selected from the group consisting of SEQ IDNOs:16, 17, 20, 21, 24 through 39, complements thereof, and fragments ofeither.

In one aspect of the present invention the nucleic acids of the presentinvention are said to be introduced nucleic acid molecules. A nucleicacid molecule is said to be “introduced” if it is inserted into a cellor organism as a result of human manipulation, no matter how indirect.Examples of introduced nucleic acid molecules include, but are notlimited to, nucleic acids that have been introduced into cells viatransformation, transfection, injection, and projection, and those thathave been introduced into an organism via methods including, but notlimited to, conjugation, endocytosis, and phagocytosis. The cell ororganism can be, or can be derived from, a plant, plant cell, algaecell, algae, fungal cell, fungus, or bacterial cell.

As used herein, “essentially unaffected” refers to a level of an agentsuch as a protein or mRNA transcript that is either not altered by aparticular event or altered only to an extent that does not affect thephysiological function of that agent. In a preferred aspect, the levelof the agent that is essentially unaffected is within 20%, morepreferably within 10%, and even more preferably within 5% of the levelat which it is found in a cell or organism that lacks a nucleic acidmolecule capable of selectively reducing another agent.

As used herein, “substantially unaffected” refers to a level of an agentsuch as a protein or mRNA transcript in which the level of the agentthat is substantially unaffected is within 49%, more preferably within35%, and even more preferably within 24% of the level at which it isfound in a cell or organism that lacks a nucleic acid molecule capableof selectively reducing another agent.

As used herein, “partially unaffected” refers to a level of an agentsuch as a protein or mRNA transcript in which the level of the agentthat is partially unaffected is within 80%, more preferably within 65%,and even more preferably within 50% of the level at which it is found ina cell or organism that lacks a nucleic acid molecule capable ofselectively reducing another agent.

As used herein, “a selective reduction” of an agent such as a protein ormRNA is relative to a cell or organism lacking a nucleic acid moleculecapable of selectively reducing the agent. In a preferred aspect, thelevel of the agent is selectively reduced by at least 50%, preferably atleast more than 75%, and even more preferably by at least more than 90%or 95%.

When levels of an agent are compared, such a comparison is preferablycarried out between organisms with a similar genetic background. In apreferred aspect, a similar genetic background is a background where theorganisms being compared share 50% or greater of their nuclear geneticmaterial. In a more preferred aspect a similar genetic background is abackground where the organisms being compared share 75% or greater, evenmore preferably 90% or greater of their nuclear genetic material. Inanother even more preferable aspect, a similar genetic background is abackground where the organisms being compared are plants, and the plantsare isogenic except for any genetic material originally introduced usingplant transformation techniques.

In an embodiment of the present invention, a nucleic acid molecule, whenintroduced into a cell or organism, is capable of selectively reducingthe level of a protein and/or transcript encoded by a FAD2 gene whileleaving the level of a protein and/or transcript encoded by a secondFAD2 gene partially unaffected, substantially unaffected, or essentiallyunaffected. In a preferred aspect, the capability of a nucleic acidmolecule to selectively reduce the level of a gene relative to anothergene is carried out by a comparison of levels of mRNA transcripts. Asused herein, mRNA transcripts include processed and non-processed mRNAtranscripts.

In another embodiment, a nucleic acid molecule, when introduced into acell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD2-1 gene while leaving thelevel of a protein and/or transcript encoded by a FAD2-2 gene partiallyunaffected, substantially unaffected, or essentially unaffected. In adifferent embodiment, a nucleic acid molecule, when introduced into acell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD2-2 gene while leaving thelevel of a protein and/or transcript encoded by a FAD2-1 gene partiallyunaffected, substantially unaffected, or essentially unaffected.

In a further embodiment, a nucleic acid molecule, when introduced into acell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD2 gene while leaving the levelof a protein and/or transcript encoded by a FAD3 gene partiallyunaffected, substantially unaffected, or essentially unaffected. In apreferred embodiment, a nucleic acid molecule, when introduced into acell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD2-1 gene while leaving thelevel of a protein and/or transcript encoded by a FAD3 gene partiallyunaffected, substantially unaffected, or essentially unaffected.

In a different embodiment, a nucleic acid molecule, when introduced intoa cell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD3 gene while leaving the levelof a protein and/or transcript encoded by another FAD3 gene partiallyunaffected, substantially unaffected, or essentially unaffected.

In an additional embodiment, a nucleic acid molecule, when introducedinto a cell or organism, is capable of selectively reducing the level ofa protein and/or transcript encoded by a FAD3-1C gene while leaving thelevel of a protein and/or transcript encoded by a FAD3-1B gene partiallyunaffected, substantially unaffected, or essentially unaffected. In anadditional embodiment, a nucleic acid molecule, when introduced into acell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD3-1C gene while leaving thelevel of a protein and/or transcript encoded by a FAD3-1A gene partiallyunaffected, substantially unaffected, or essentially unaffected.

In a different embodiment, a nucleic acid molecule, when introduced intoa cell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD3-1B gene while leaving thelevel of a protein and/or transcript encoded by a FAD3-1C gene partiallyunaffected, substantially unaffected, or essentially unaffected. In adifferent embodiment, a nucleic acid molecule, when introduced into acell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD3-1B gene while leaving thelevel of a protein and/or transcript encoded by a FAD3-1A gene partiallyunaffected, substantially unaffected, or essentially unaffected.

In a further embodiment, a nucleic acid molecule, when introduced into acell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD3-1A gene while leaving thelevel of a protein and/or transcript encoded by a FAD3-1B gene partiallyunaffected, substantially unaffected, or essentially unaffected. In anadditional embodiment, a nucleic acid molecule, when introduced into acell or organism, is capable of selectively reducing the level of aprotein and/or transcript encoded by a FAD3-1A gene while leaving thelevel of a protein and/or transcript encoded by a FAD3-1C gene partiallyunaffected, substantially unaffected, or essentially unaffected.

Further preferred embodiments of the invention are nucleic acidmolecules that are at least 50%, 60%, or 70% identical over their entirelength to a nucleic acid molecule of the invention, and nucleic acidmolecules that are complementary to such nucleic acid molecules. Morepreferable are nucleic acid molecules that comprise a region that is atleast 80% or 85% identical over its entire length to a nucleic acidmolecule of the invention and nucleic acid molecules that arecomplementary thereto. In this regard, nucleic acid molecules at least90% identical over their entire length are particularly preferred, thoseat least 95% identical are especially preferred. Further, those with atleast 97% identity are highly preferred and those with at least 98% and99% identity are particularly highly preferred, with those at least 99%being the most highly preferred.

The invention also provides a nucleic acid molecule comprising a nucleicacia molecule sequence obtainable by screening an appropriate librarycontaining the complete gene for a nucleic acid molecule sequence setforth in the Sequence Listing under stringent hybridization conditionswith a probe having the sequence of said nucleic acid molecule sequenceor a fragment thereof; and isolating said nucleic acid moleculesequence. Fragments useful for obtaining such a nucleic acid moleculeinclude, for example, probes and primers as described herein.

Nucleic acid molecules of the invention can be used as a hybridizationprobe for RNA, cDNA, or genomic DNA to isolate full length cDNAs orgenomic clones and to isolate cDNA or genomic clones of other genes thathave a high sequence similarity to the sequence of a nucleic acidmolecule set forth in the Sequence Listing.

The nucleic acid molecules of the present invention can be readilyobtained by using the herein described nucleic acid molecules orfragments thereof to screen cDNA or genomic libraries obtained fromplant species or other appropriate organisms. These methods are known tothose of skill in the art, as are methods for forming such libraries. Inone embodiment, such sequences are obtained by incubating nucleic acidmolecules of the present invention with members of genomic libraries andrecovering clones that hybridize to such nucleic acid molecules thereof.In a second embodiment, methods of chromosome walking or inverse PCR maybe used to obtain such sequences. In a third embodiment, the sequence ofa nucleic acid molecule of the present invention may be used to screen alibrary or database, using bioinformatics techniques known in the art.See, e.g., Bioinformatics, Baxevanis & Ouellette, eds.,Wiley-Interscience (1998).

Any of a variety of methods may be used to obtain one or more of thenucleic acid molecules of the present invention. Automated nucleic acidsynthesizers may be employed for this purpose, and to make a nucleicacid molecule that has a sequence also found in a cell or organism. Inlieu of such synthesis, the disclosed nucleic acid molecules may be usedto define a pair of primers that can be used with the polymerase chainreaction to amplify and obtain any desired nucleic acid molecule orfragment.

“Identity,” as is well understood in the art, is a relationship betweentwo or more polypeptide sequences or two or more nucleic acid moleculesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or nucleic acid molecule sequences, as determined by thematch between strings of such sequences. “Identity” can be readilycalculated by known methods including, but not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York (1988); Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M. and Griffin, H. G.,eds., Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press (1987); Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York(1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math, 48:1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available programs. Computer programswhich can be used to determine identity between two sequences include,but are not limited to, GCG (Devereux, J., et al., Nucleic AcidsResearch 12(1):387 (1984); suite of five BLAST programs, three designedfor nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and twodesigned for protein sequence queries (BLASTP and TBLASTN) (Coulson,Trends in Biotechnology, 12:76-80 (1994); Birren et al., GenomeAnalysis, 1:543-559 (1997)). The BLASTX program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH, Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol.,215:403-410 (1990)). The well-known Smith Waterman algorithm can also beused to determine identity.

Parameters for polypeptide sequence comparison typically include thefollowing:

Algorithm: Needleman and Wunsch, J. Mol. Biol., 48:443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA, 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program which can be used with these parameters is publicly availableas the “gap” program from Genetics Computer Group, Madison, Wis. Theabove parameters along with no penalty for end gap are the defaultparameters for peptide comparisons.

-   -   Parameters for nucleic acid molecule sequence comparison include        the following:    -   Algorithm: Needleman and Wunsch, J. Mol. Bio., 48:443-453 (1970)    -   Comparison matrix: matches—+10; mismatches=0    -   Gap Penalty: 50    -   Gap Length Penalty: 3

As used herein, “% identity” is determined using the above parameters asthe default parameters for nucleic acid molecule sequence comparisonsand the “gap” program from GCG, version 10.2.

The invention further relates to nucleic acid molecules that hybridizeto nucleic acid molecules of the present invention. In particular, theinvention relates to nucleic acid molecules that hybridize understringent conditions to the above-described nucleic acid molecules. Asused herein, the terms “stringent conditions” and “stringenthybridization conditions” mean that hybridization will generally occurif there is at least 95% and preferably at least 97% identity betweenthe sequences. An example of stringent hybridization conditions isovernight incubation at 42° C. in a solution comprising 50% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20micrograms/milliliter denatured, sheared salmon sperm DNA, followed bywashing the hybridization support in 0.1×SSC at approximately 65° C.Other hybridization and wash conditions are well known and areexemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y. (1989), particularly Chapter11.

In embodiments where nucleic acid sequences which when expressed arecapable of selectively reducing the level of a protein and/or transcriptencoded by a FAD2-1 gene and the level of a protein and/or transcriptencoded by at least one FAD3 gene while leaving the level of a proteinand/or transcript of a FAD2-2 gene in the plant partially, substantiallyor essentially unaffected, preferred FAD2-1 nucleic acid sequences areselected from the groups consisting of (1) nucleic acid sequences withat least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequenceidentity over the entire length of the nucleic acid molecule with anucleotide sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2 and fragments thereof, where such a nucleic acid sequencedoes not hybridize under stringent conditions to a nucleic acid moleculewith a nucleotide sequence of SEQ ID NO:4; (2) nucleic acid moleculeswhich contain sequences that are also found in a soybean FAD2-1 geneintron; and (3) nucleic acid molecules that exhibit sequences with atleast 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequenceidentity over the entire length of the nucleic acid molecule with anucleic acid molecule of (2).

In embodiments where nucleic acid sequences which when expressed arecapable of selectively reducing the level of a protein and/or transcriptencoded by a FAD2-2 gene and the level of a protein and/or transcriptencoded by at least one FAD3 gene while leaving the level of a proteinand/or transcript of a FAD2-1 gene in the plant partially, substantiallyor essentially unaffected, preferred FAD2-2 nucleic acid sequences areselected from the groups consisting of (1) nucleic acid sequences withat least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequenceidentity over the entire length of the nucleic acid molecule with anucleotide sequence of SEQ ID NO: 4 and fragments thereof, where such anucleic acid sequence does not hybridize under stringent conditions to anucleic acid molecule with a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2; (2) nucleic acid moleculeswhich contain sequences that are also found in a soybean FAD2-2 geneintron; and (3) nucleic acid molecules that exhibit sequences with atleast 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequenceidentity over the entire length of the nucleic acid molecule with anucleic acid molecule of (2).

In embodiments where nucleic acid sequences which when expressed arecapable of selectively reducing a FAD3 gene, preferred FAD3 nucleic acidsequences are selected from the groups consisting of (1) nucleic acidsequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or100% sequence identity over the entire length of the nucleic acidmolecule with a nucleotide sequence selected from the group consistingof SEQ ID NOs:5-14, and fragments thereof, where such a nucleic acidsequence does not hybridize under stringent conditions to a nucleic acidmolecule with a nucleotide sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4; (2) nucleic acid moleculeswhich contain sequences that are also found in a soybean FAD3 geneintron; and (3) nucleic acid molecules that exhibit sequences with atleast 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequenceidentity over the entire length of the nucleic acid molecule with anucleic acid molecule of (2).

One subset of the nucleic acid molecules of the invention includesfragment nucleic acid molecules. Fragment nucleic acid molecules mayconsist of significant portion(s) of, or indeed most of, the nucleicacid molecules of the invention, such as those specifically disclosed.Alternatively, the fragments may comprise smaller oligonucleotides(having from about 15 to about 400 contiguous nucleotide residues andmore preferably, about 15 to about 30 contiguous nucleotide residues, orabout 50 to about 100 contiguous nucleotide residues, or about 100 toabout 200 contiguous nucleotide residues, or about 200 to about 400contiguous nucleotide residues, or about 275 to about 350 contiguousnucleotide residues).

In another aspect, a fragment nucleic acid molecule has a nucleic acidsequence that is at least 15, 25, 50, or 100 contiguous nucleotides of anucleic acid molecule of the present invention. In a preferredembodiment, the nucleic acid molecule has a nucleic acid sequence thatis at least 15, 25, 50, or 100 contiguous nucleotides of a nucleic acidmolecule having a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1 through SEQ ID NO:14 and complements thereof.

A fragment of one or more of the nucleic acid molecules of the presentinvention may be a probe and specifically a PCR probe. A PCR probe is anucleic acid molecule capable of initiating a polymerase activity whilein a double-stranded structure with another nucleic acid molecule.Various methods for determining the structure of PCR probes and PCRtechniques exist in the art. Computer generated searches using programssuch as Primer3 (www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi),STSPipeline (www-genome.wi.mit.edu/cgi-bin/www-STS_Pipeline), or GeneUp(Pesole et al., BioTechniques 25:112-123 (1998)), for example, can beused to identify potential PCR primers.

Nucleic acid molecules or fragments thereof of the present invention arecapable of specifically hybridizing to other nucleic acid moleculesunder certain circumstances. Nucleic acid molecules of the presentinvention include those that specifically hybridize to nucleic acidmolecules having a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1 through SEQ ID NO:14, complements thereof, andfragments of either.

As used herein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure.

A nucleic acid molecule of the invention can also encode a homolognucleic acid molecule. As used herein, a homolog nucleic acid moleculeor fragment thereof is a counterpart nucleic acid molecule or fragmentthereof in a second species (e.g., corn FAD2-1 intron nucleic acidmolecule is a homolog of Arabidopsis FAD2-1 intron nucleic acidmolecule). A homolog can also be generated by molecular evolution or DNAshuffling techniques, so that the molecule retains at least onefunctional or structure characteristic of the original polypeptide (see,for example, U.S. Pat. No. 5,811,238).

In another embodiment, the homolog is obtained from a plant selectedfrom the group consisting of alfalfa, Arabidopsis, barley, Brassicacampestris, oilseed rape, broccoli, cabbage, canola, citrus, cotton,garlic, oat, Allium, flax, an ornamental plant, jojoba, corn, peanut,pepper, potato, rapeseed, rice, rye, sorghum, strawberry, sugarcane,sugarbeet, tomato, wheat, poplar, pine, fir, eucalyptus, apple, lettuce,lentils, grape, banana, tea, turf grasses, sunflower, Phaseolus, crambe,mustard, castor bean, sesame, cottonseed, linseed, safflower, and oilpalm. More particularly, a preferred homolog is obtained from a plantselected from the group consisting of canola, corn, Brassica campestris,oilseed rape, soybean, crambe, mustard, castor bean, peanut, sesame,cottonseed, linseed, rapeseed, safflower, oil palm, flax, and sunflower.In an even more preferred embodiment, the homolog is obtained from aplant selected from the group consisting of canola, rapeseed, corn,Brassica campestris, oilseed rape, soybean, sunflower, safflower, oilpalm, and peanut.

Plant Constructs and Plant Transformants

One or more of the nucleic acid molecules of the invention may be usedin plant transformation or transfection. Exogenous genetic material maybe transferred into a plant cell and the plant cell regenerated into awhole, fertile or sterile plant or plant part. Exogenous geneticmaterial is any genetic material, whether naturally occurring orotherwise, from any source that is capable of being inserted into anyorganism.

A plant can have a family of more than one FAD2 or FAD3 genes (i.e.,genes which encode an enzyme with the specified activity present atdifferent locations within the genome of the plants). As used herein, a“FAD2 gene family member” is any FAD2 gene found within the geneticmaterial of the plant. As used herein, a “FAD3 gene family member” isany FAD3 gene found within the genetic material of the plant. In oneembodiment, a gene family can be additionally classified by thesimilarity of the nucleic acid sequences. In a preferred aspect of thisembodiment, a gene family member exhibits at least 60%, more preferablyat least 70%, more preferably at least 80% nucleic acid sequenceidentity in the coding sequence portion of the gene.

In one aspect of the present invention, a plant contains adouble-stranded RNAi construct where a first promoter is operably linkedto a first nucleic acid molecule, having a first nucleic acid sequencethat has at least about 85% identity to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:1 through SEQ ID NO:2,complements thereof, and fragments of either, and a second nucleic acidmolecule that has a second nucleic acid sequence with at least about 85%identity to a nucleic acid sequence selected from the group consistingof SEQ ID NO:4 through SEQ ID NO:14, complements thereof, and fragmentsof either, is operably linked to the first nucleic acid molecule.

Also provided by the present invention is a plant comprising adouble-stranded RNAi construct where a first promoter is operably linkedto a first nucleic acid molecule, having a first nucleic acid sequencethat has at least about 85% identity to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:1 through SEQ ID NO:2,complements thereof, and fragments of either, wherein a second nucleicacid molecule having a second nucleic acid sequence that has at leastabout 85% identity to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:4 through SEQ ID NO:14, complements thereof, andfragments of either is operably linked to a second promoter in a dsRNAiconfiguration.

In one embodiment of the invention, the expression level of a protein ortranscript in one family member of that gene is selectively reducedwhile leaving the level of a protein or transcript of a second familymember partially unaffected. In a preferred embodiment of the invention,the expression level of a protein or transcript in one family member ofthat gene is selectively reduced while leaving the level of a protein ortranscript of a second family member substantially unaffected. In ahighly preferred embodiment of the invention, the expression level of aprotein or transcript in one family member of that gene is selectivelyreduced while leaving the level of a protein or transcript of a secondfamily member essentially unaffected.

In a particularly preferred embodiment, a plant of the present inventionincludes nucleic acid sequences which when expressed are capable ofselectively reducing the expression level of a protein and/or transcriptencoded by certain FAD2 and FAD3 genes while leaving the level of aprotein and/or transcript of at least one other FAD2 or FAD3 gene in theplant partially unaffected. In a particularly preferred embodiment, aplant of the present invention includes nucleic acid sequences whichwhen expressed are capable of selectively reducing the expression levelof a protein and/or transcript encoded by certain FAD2 and FAD3 geneswhile leaving the level of a protein and/or transcript of at least oneother FAD2 or FAD3 gene in the plant substantially unaffected. In aparticularly preferred embodiment, a plant of the present inventionincludes nucleic acid sequences which when expressed are capable ofselectively reducing the expression level of a protein and/or transcriptencoded by certain FAD2 and FAD3 genes while leaving the level of aprotein and/or transcript of at least one other FAD2 or FAD3 gene in theplant essentially unaffected.

In a more particularly preferred embodiment, a soybean plant of thepresent invention includes nucleic acid sequences which when expressedare capable of selectively reducing the expression level of a proteinand/or transcript encoded by a FAD2-1 gene and at least one FAD3 genewhile leaving the level of a protein and/or transcript of a FAD2-2 genein the plant partially unaffected. In a more particularly preferredembodiment, a soybean plant of the present invention includes nucleicacid sequences which when expressed are capable of selectively reducingthe expression level of a protein and/or transcript encoded by a FAD2-1gene and at least one FAD3 gene while leaving the level of a proteinand/or transcript of a FAD2-2 gene in the plant substantiallyunaffected. In a more particularly preferred embodiment, a soybean plantof the present invention includes nucleic acid sequences which whenexpressed are capable of selectively reducing the expression level of aprotein and/or transcript encoded by a FAD2-1 gene and at least one FAD3gene while leaving the level of a protein and/or transcript of a FAD2-2gene in the plant essentially unaffected.

In another more particularly preferred embodiment, a soybean plant ofthe present invention includes nucleic acid sequences which whenexpressed are capable of selectively reducing the expression level of aprotein and/or transcript encoded by a FAD2-1 gene and at least two,three or more FAD3 genes while leaving the level of a protein and/ortranscript of a FAD2-2 gene in the plant partially unaffected. Inanother more particularly preferred embodiment, a soybean plant of thepresent invention includes nucleic acid sequences which when expressedare capable of selectively reducing the expression level of a proteinand/or transcript encoded by a FAD2-1 gene and at least two, three ormore FAD3 genes while leaving the level of a protein and/or transcriptof a FAD2-2 gene in the plant substantially unaffected. In another moreparticularly preferred embodiment, a soybean plant of the presentinvention includes nucleic acid sequences which when expressed arecapable of selectively reducing the expression level of a protein and/ortranscript encoded by a FAD2-1 gene and at least two, three or more FAD3genes while leaving the level of a protein and/or transcript of a FAD2-2gene in the plant essentially unaffected.

In a preferred embodiment, a soybean of the present invention includesexogenous nucleic acid sequences selected from the groups consisting ofa FAD3 intron or fragment thereof, more preferably from a nucleic acidmolecule selected from the group consisting of SEQ ID NOs:5-14, orfragments thereof.

In a particularly preferred embodiment, a soybean of the presentinvention includes a nucleic acid sequence which when expressed iscapable of reducing the expression level of a protein and/or transcriptencoded by a FAD3-1C gene while leaving the level of a protein and/ortranscript of a FAD3-1B gene in the plant partially unaffected. In aparticularly preferred embodiment, a soybean of the present inventionincludes a nucleic acid sequence which when expressed is capable ofreducing the expression level of a protein and/or transcript encoded bya FAD3-1C gene while leaving the level of a protein and/or transcript ofa FAD3-1B gene in the plant substantially unaffected. In a particularlypreferred embodiment, a soybean of the present invention includes anucleic acid sequence which when expressed is capable of reducing theexpression level of a protein and/or transcript encoded by a FAD3-1Cgene while leaving the level of a protein and/or transcript of a FAD3-1Bgene in the plant essentially unaffected.

In embodiments where nucleic acid sequences which when expressed arecapable of selectively reducing the expression level of a protein and/ortranscript encoded by a FAD2-1 gene and at least one FAD3 genes whileleaving the level of a protein and/or transcript of a FAD2-2 gene in theplant partially unaffected, substantially unaffected, or essentiallyunaffected, preferred FAD2-1 nucleic acid sequences are selected fromthe groups consisting of (1) nucleic acid sequences with at least 50%,60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity overthe entire length of the nucleic acid molecule with a nucleotidesequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2and fragments thereof, where such a nucleic acid sequence does nothybridize under stringent conditions to a nucleic acid molecule with anucleotide sequence of SEQ ID NO:4; (2) nucleic acid molecules whichcontain sequences that are also found in a soybean FAD2-1 gene intron;and (3) nucleic acid molecules that exhibit sequences with at least 50%,60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity overthe entire length of the nucleic acid molecule with a nucleic acidmolecule of (2).

In embodiments where nucleic acid sequences which when expressed arecapable of selectively reducing the expression level of a protein and/ortranscript encoded by a FAD3 gene, preferred FAD3 nucleic acid sequencesare selected from the groups consisting of (1) nucleic acid sequenceswith at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100%sequence identity over the entire length of the nucleic acid moleculewith a nucleotide sequence selected from the group consisting of SEQ IDNOs:5-14, and fragments thereof, where such a nucleic acid sequence doesnot hybridize under stringent conditions to a nucleic acid molecule witha nucleotide sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, and SEQ ID NO:4; (2) nucleic acid molecules which containsequences that are also found in a soybean FAD3 gene intron; and (3)nucleic acid molecules that exhibit sequences with at least 50%, 60%,70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity over theentire length of the nucleic acid molecule with a nucleic acid moleculeof (2).

In a preferred embodiment, a soybean seed of the present invention hasan oil composition that is 50% or greater oleic acid and 10% or lesslinolenic acid. In a more preferred embodiment, a soybean seed of thepresent invention has an oil composition that is 60% or greater oleicacid and 7% or less linolenic acid. In a particular preferredembodiment, a soybean seed of the present invention has an oilcomposition that is 65% or greater oleic acid and 5% or less linolenicacid, preferably 4% or less linolenic acid, and more preferably 3% orless linolenic acid. As used herein, all % composition of oils within aplant or plant part such as a seed are determined by relative molepercent.

In another preferred embodiment a soybean seed of the present inventionhas an oil composition that is between 50% and 90% oleic acid, and 10%or less linolenic acid. In a more preferred embodiment, a soybean seedof the present invention has an oil composition that is between 60% and80% oleic acid, and 7% or less linolenic acid. In a particular preferredembodiment, a soybean seed of the present invention has an oilcomposition that is between 65% and 75% oleic acid, and 5% or lesslinolenic acid, preferably 4% or less linolenic acid, and morepreferably 3% or less linolenic acid.

In another preferred embodiment a soybean seed of the present inventionhas an oil composition that is between 50% and 90% oleic acid, between8% and 16% palmitic acid and 10% or less linolenic acid. In a morepreferred embodiment, a soybean seed of the present invention has an oilcomposition that is between 60% and 80% oleic acid, between 6% and 12%palmitic acid and 7% or less linolenic acid. In a particularly preferredembodiment, a soybean seed of the present invention has an oilcomposition that is between 65% and 75% oleic acid, between 8% and 11%palmitic acid and 5% or less linolenic acid, preferably 4% or lesslinolenic acid, and more preferably 3% or less linolenic acid.

In a particularly preferred embodiment, a soybean seed of the presentinvention has an oil composition that is between 65% and 75% oleic acid,and 5% or less linolenic acid, preferably 4% or less linolenic acid, andmore preferably 3% or less linolenic acid, where nucleic acid sequenceswhich when expressed are capable of selectively reducing the expressionlevel of a protein and/or transcript encoded by a FAD2-1 gene and atleast one FAD3 gene while leaving the level of a protein and/ortranscript of a FAD2-2 gene in the plant partially unaffected,substantially unaffected, or essentially unaffected, the FAD2-1 nucleicacid sequences are selected from the groups consisting of: (1) nucleicacid sequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%or 100% sequence identity over the entire length of the nucleic acidmolecule with a nucleotide sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:2 and fragments thereof, where such a nucleicacid sequence does not hybridize under stringent conditions to a nucleicacid molecule with a nucleotide sequence of SEQ ID NO:4; (2) nucleicacid molecules which contain sequences that are also found in a soybeanFAD2-1 gene intron; and (3) nucleic acid molecules that exhibitsequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or100% sequence identity over the entire length of the nucleic acidmolecule with a nucleic acid molecule of (2); and the FAD3 nucleic acidsequences are selected from the groups consisting of (1) nucleic acidsequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or100% sequence identity over the entire length of the nucleic acidmolecule with a nucleotide sequence selected from the group consistingof SEQ ID NOs:5-14, and fragments thereof, where such a nucleic acidsequence does not hybridize under stringent conditions to a nucleic acidmolecule with a nucleotide sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:4; (2) nucleic acid moleculeswhich contain sequences that are also found in a soybean FAD3 geneintron; and (3) nucleic acid molecules that exhibit sequences with atleast 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequenceidentity over the entire length of the nucleic acid molecule with anucleic acid molecule of (2).

In another embodiment, a soybean seed of the present invention has anoil composition that is 80% or greater, more preferably 90% or greateroleic acid and 5% or less linolenic acid, preferably 4% or lesslinolenic acid, and more preferably 3% or less linolenic acid.

In a preferred embodiment, a soybean seed of the present invention hasan oil composition that is 80% or greater, more preferably 90% orgreater oleic acid and 5% or less linolenic acid, preferably 4% or lesslinolenic acid, and more preferably 3% or less linolenic acid, where thenucleic acid sequences are capable of reducing the expression of FAD2-1,FAD2-2 and at least one FAD3 genes. In a particularly preferredembodiment of this aspect, the nucleic acid sequences are selected fromthe group consisting of SEQ ID NO:1 through SEQ ID NO:14, complementsthereof, and fragments of either.

In a preferred embodiment of the present invention, a soybean seed ofthe present invention has an oil composition of 50% or greater oleicacid, more preferably 60% or greater, 70% or greater, 80% or greater, or90% or greater oleic acid.

In another preferred embodiment of the present invention, a soybean seedof the present invention has an oil composition that is 10% or lesslinolenic acid, more preferably 5% or less, 4% or less, or 3% or lesslinolenic acid.

Analogous genetic material may also be obtained from other species, forexample monocotyledons or dicotyledons, including, but not limited tocanola, corn, soybean, Arabidopsis, Phaseolus, peanut, alfalfa, wheat,rice, oat, sorghum, rapeseed, rye, barley, millet, fescue, perennialryegrass, sugarcane, cranberry, papaya, banana, safflower, oil palm,flax, muskmelon, apple, cucumber, dendrobium, gladiolus, chrysanthemum,liliacea, cotton, eucalyptus, sunflower, Brassica campestris, oilseedrape, turfgrass, sugarbeet, coffee and dioscorea (Christou, INO:Particle Bombardment Genetic Engineering of Plants, BiotechnologyIntelligence Unit. Academic Press, San Diego, Calif. (1996)), withcanola, corn, Brassica campestris, oilseed rape, rapeseed, soybean,crambe, mustard, castor bean, peanut, sesame, cottonseed, linseed,safflower, oil palm, flax, and sunflower preferred, and canola,rapeseed, corn, Brassica campestris, oilseed rape, soybean, sunflower,safflower, oil palms, and peanut more preferred. In a more preferredembodiment, the genetic material is transferred into canola. In anothermore preferred embodiment, the genetic material is transferred intooilseed rape. In another particularly preferred embodiment, the geneticmaterial is transferred into soybean.

The levels of products such as transcripts or proteins may be increasedor decreased throughout an organism such as a plant or localized in oneor more specific organs or tissues of the organism. For example thelevels of products may be increased or decreased in one or more of thetissues and organs of a plant including without limitation: roots,tubers, stems, leaves, stalks, fruit, berries, nuts, bark, pods, seedsand flowers. A preferred organ is a seed.

Exogenous genetic material may be transferred into a host cell by theuse ot a DNA vector or construct designed for such a purpose. Design ofsuch a vector is generally within the skill of the art (See, PlantMolecular Biology: A Laboratory Manual, Clark (ed.), Springer, N.Y.(1997)).

A construct or vector may include a plant promoter to express thenucleic acid molecule of choice. In a preferred embodiment, any nucleicacid molecules described herein can be operably linked to a promoterregion which functions in a plant cell to cause the production of anmRNA molecule. For example, any promoter that functions in a plant cellto cause the production of an mRNA molecule, such as those promotersdescribed herein, without limitation, can be used. In a preferredembodiment, the promoter is a plant promoter.

A number of promoters that are active in plant cells have been describedin the literature. These include, but are not limited to, the nopalinesynthase (NOS) promoter (Ebert et al., Proc. Natl. Acad. Sci. (U.S.A.)84:5745-5749 (1987)), the octopine synthase (OCS) promoter (which iscarried on tumor-inducing plasmids of Agrobacterium turnefaciens), thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Spromoter (Lawton et al., Plant Mol. Biol.9:315-324 (1987)) and the CaMV35S promoter (Odell et al., Nature 313:810-812 (1985)), the figwortmosaic virus 35S-promoter (U.S. Pat. No. 5,378,619), the light-induciblepromoter from the small subunit of ribulose-1,5-bis-phosphatecarboxylase (ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl.Acad. Sci. (U.S.A.) 84:6624-6628 (1987)), the sucrose synthase promoter(Yang et al., Proc. Natl. Acad. Sci. (U.S.A) 87:4144-4148 (1990)), the Rgene complex promoter (Chandler et al., The Plant Cell 1:1175-1183(1989)) and the chlorophyll a/b binding protein gene promoter. Thesepromoters have been used to create DNA constructs that have beenexpressed in plants; see, e.g., PCT publication WO 84/02913. The CaMV35S promoters are preferred for use in plants. Promoters known or foundto cause transcription of DNA in plant cells can be used in theinvention.

Particularly preferred promoters can also be used to express a nucleicacid molecule of the present invention in seeds or fruits. Indeed, in apreferred embodiment, the promoter used is a seed specific promoter.Examples of such promoters include the 5′ regulatory regions from suchgenes as napin (Kridl et al., Seed Sci. Res. 1:209:219 (1991)),phaseolin (Bustos, et al., Plant Cell, 1(9):839-853 (1989)), soybeantrypsin inhibitor (Riggs, et al., Plant Cell 1( 6 ):609-621 (1989)), ACP(Baerson, et al., Plant Mol. Biol., 22(2):255-267 (1993)), stearoyl-ACPdesaturase (Slocombe, et al., Plant Physiol. 104(4):167-176 (1994)),soybean a subunit of b-conglycinin (soy 7s, (Chen et al., Proc. Natl.Acad. Sci., 83:8560-8564 (1986))), and oleosin (see, for example, Hong,et al., Plant Mol. Biol., 34(3):549-555 (1997)). Further examplesinclude the promoter for β-conglycinin (Chen et al., Dev. Genet.10:112-122 (1989)) and the promoter for FAE (PCT Publication WO01/11061). Preferred promoters for expression in the seed are 7S andnapin promoters.

Additional promoters that may be utilized are described, for example, inU.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147; 5,447,858; 5,608,144;5,608,144; 5,614,399; 5,633,441; 5,633,435; and 4,633,436. In addition,a tissue specific enhancer may be used (Fromm et al., The Plant Cell1:977-984 (1989)).

Constructs or vectors may also include, with the region of interest, anucleic acid sequence that acts, in whole or in part, to terminatetranscription of that region. A number of such sequences have beenisolated, including the Tr7 3′ sequence and the NOS 3′ sequence(Ingelbrecht et al., The Plant Cell 1:671-680 (1989); Bevan et al.,Nucleic Acids Res. 11:369-385 (1983)). Regulatory transcript terminationregions can be provided in plant expression constructs of this inventionas well. Transcript termination regions can be provided by the DNAsequence encoding the gene of interest or a convenient transcriptiontermination region derived from a different gene source, for example,the transcript termination region that is naturally associated with thetranscript initiation region. The skilled artisan will recognize thatany convenient transcript termination region that is capable ofterminating transcription in a plant cell can be employed in theconstructs of the present invention.

A vector or construct may also include regulatory elements. Examples ofsuch include the Adh intron 1 (Callis et al., Genes and Develop.1:1183-1200 (1987)), the sucrose synthase intron (Vasil et al., PlantPhysiol. 91:1575-1579 (1989)) and the TMV omega element (Gallie et al.,The Plant Cell 1:301-311 (1989)). These and other regulatory elementsmay be included when appropriate.

A vector or construct may also include a selectable marker. Selectablemarkers may also be used to select for plants or plant cells thatcontain the exogenous genetic material. Examples of such include, butare not limited to: a neo gene (Potrykus et al., Mol. Gen. Genet.199:183-188 (1985)), which codes for kanamycin resistance and can beselected for using kanamycin, RptII, G418, hpt; a bar gene which codesfor bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al.,Bio/Technology 6:915-922 (1988); Reynaerts et al., Selectable andScreenable Markers. In Gelvin and Schilperoort. Plant Molecular BiologyManual, Kluwer, Dordrecht (1988); Reynaerts et al., Selectable andscreenable markers. In Gelvin and Schilperoort. Plant Molecular BiologyManual, Kluwer, Dordrecht (1988)), aadA (Jones et al., Mol. Gen. Genet.(1987)),) which encodes glyphosate resistance; a nitrilase gene whichconfers resistance to bromoxynil (Stalker et al., J. Biol. Chem.263:6310-6314 (1988)); a mutant acetolactate synthase gene (ALS) whichconfers imidazolinone or sulphonylurea resistance (European PatentApplication 154,204 (Sep. 11, 1985)), ALS (D'Halluin et al.,Bio/Technology 10:309-314 (1992)), and a methotrexate resistant DHFRgene (Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)).

A vector or construct may also include a screenable marker. Screenablemarkers may be used to monitor expression. Exemplary screenable markersinclude: a β-glucuronidase or uidA gene (GUS) which encodes an enzymefor which various chromogenic substrates are known (Jefferson, PlantMol. Biol, Rep. 5:387-405 (1987); Jefferson et al., EMBO J. 6:3901-3907(1987)); an R-locus gene, which encodes a product that regulates theproduction of anthocyanin pigments (red color) in plant tissues(Dellaporta et al., Stadler Symposium 11:263-282 (1988)); a β-lactamasegene (Sutcliffe et al., Proc. Natl. Acad. Sci. (U.S.A.) 75:3737-3741(1978)), a gene which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); aluciferase gene (Ow et al., Science 234:856-859 (1986)); a xylE gene(Zukowsky et al., Proc. Natl. Acad. Sci. (U.S.A) 80:1101-1105 (1983))which encodes a catechol dioxygenase that can convert chromogeniccatechols; an (α-amylase gene (Ikatu et al., Bio/Technol. 8:241-242(1990)); a tyrosinase gene (Katz et al., J. Gen. Microbiol.129:2703-2714 (1983)) which encodes an enzyme capable of oxidizingtyrosine to DOPA and dopaquinone which in turn condenses to melanin; anα-galactosidase, which will turn a chromogenic (α-galactose substrate.

Included within the terms “selectable or screenable marker genes” arealso genes that encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers that encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes that canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins that are detectable,(e.g., by ELISA), small active enzymes that are detectable inextracellular solution (e.g., α-amylase, β-lactamase, phosphinothricintransferase), or proteins that are inserted or trapped in the cell wall(such as proteins that include a leader sequence such as that found inthe expression unit of extension or tobacco PR-S). Other possibleselectable and/or screenable marker genes will be apparent to those ofskill in the art.

It is understood that two or more nucleic molecules of the presentinvention may be introduced into a plant using a single construct andthat construct can contain more than one promoter. In embodiments wherethe construct is designed to express two nucleic acid molecules, it ispreferred that the two promoters are (i) two constitutive promoters,(ii) two seed-specific promoters, or (iii) one constitutive promoter andone seed-specific promoter. Preferred seed-specific and constitutivepromoters are a napin and a CaMV promoter, respectively. Illustrativecombinations are set forth in Example 8. It is understood that two ormore of the nucleic molecules may be physically linked and expressedutilizing a single promoter, preferably a seed-specific or constitutivepromoter.

It is further understood that two or more nucleic acids of the presentinvention may be introduced into a plant using two or more differentconstructs. Alternatively, two or more nucleic acids of the presentinvention may be introduced into two different plants and the plants maybe crossed to generate a single plant expressing two or more nucleicacids. In an RNAi embodiment, it is understood that the sense andantisense strands may be introduced into the same plant on one constructor two constructs. Alternatively, the sense and antisense strands may beintroduced into two different plants and the plants may be crossed togenerate a single plant expressing both sense and antisense strands.

Any of the nucleic acid molecules and constructs of the invention may beintroduced into a plant or plant cell in a permanent or transientmanner. Preferred nucleic acid molecules and constructs of the presentinvention are described above in the Detailed Description, and in theExamples. Another embodiment of the invention is directed to a method ofproducing transgenic plants which generally comprises the steps ofselecting a suitable plant or plant cell, transforming the plant orplant cell with a recombinant vector, and obtaining a transformed hostcell.

In a preferred embodiment the plant or cell is, or is derived from, aplant involved in the production of vegetable oils for edible andindustrial uses. Especially preferred are temperate oilseed crops.Plants of interest include, but are not limited to, rapeseed (canola andHigh Erucic Acid varieties), maize, soybean, crambe, mustard, castorbean, peanut, sesame, cotton, linseed, safflower, oil palm, flax,sunflower, and coconut. The invention is applicable to monocotyledonousor dicotyledonous species alike, and will be readily applicable to newand/or improved transformation and regulatory techniques.

Methods and technology for introduction of DNA into plant cells are wellknown to those of skill in the art, and virtually any method by whichnucleic acid molecules may be introduced into a cell is suitable for usein the present invention. Non-limiting examples of suitable methodsinclude: chemical methods; physical methods such as microinjection,electroporation, the gene gun, microprojectile bombardment, and vacuuminfiltration; viral vectors; and receptor-mediated mechanisms. Othermethods of cell transformation can also be used and include but are notlimited to introduction of DNA into plants by direct DNA transfer intopollen, by direct injection of DNA into reproductive organs of a plant,or by direct injection of DNA into the cells of immature embryosfollowed by the rehydration of desiccated embryos.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells. See, e.g., Fraley et al.,Bio/Technology 3:629-635 (1985); Rogers et al., Methods Enzymol.153:253-277 (1987). The region of DNA to be transferred is defined bythe border sequences and intervening DNA is usually inserted into theplant genome. Spielmann et al., Mol. Gen. Genet. 205:34 (1986). ModemAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations.Klee et al., In: Plant DNA Infectious Agents, Hohn and Schell (eds.),Springer-Verlag, N. Y., pp. 179-203 (1985).

The regeneration, development and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art. See generally, Maliga et al., Methods in PlantMolecular Biology, Cold Spring Harbor Press (1995); Weissbach andWeissbach, In: Methods for Plant Molecular Biology, Academic Press, SanDiego, Calif. (1988). Plants of the present invention can be part of orgenerated from a breeding program, and may also be reproduced usingapomixis. Methods for the production of apomictic plants are known inthe art. See, e.g., U.S. Pat. No. 5,811,636.

Cosuppression is the reduction in expression levels, usually at thelevel of RNA, of a particular endogenous gene or gene family by theexpression of a homologous sense construct that is capable oftranscribing mRNA of the same strandedness as the transcript of theendogenous gene (Napoli et al., Plant Cell 2:279-289 (1990); van derKrol et al., Plant Cell 2:291-299 (1990)). Cosuppression may result fromstable transformation with a single copy nucleic acid molecule that ishomologous to a nucleic acid sequence found with the cell (Prolls andMeyer, Plant J. 2:465-475 (1992)) or with multiple copies of a nucleicacid molecule that is homologous to a nucleic acid sequence found withthe cell (Mittlesten et al., Mol. Gen. Genet. 244:325-330 (1994)).Genes, even though different, linked to homologous promoters may resultin the cosuppression of the linked genes (Vaucheret, C.R. Acad. Sci. III316:1471-1483 (1993); Flavell, Proc. Natl. Acad. Sci. (U.S.A.)91:3490-3496 (1994)); van Blokland et al, Plant J. 6:861-877 (1994);Jorgensen, Trends Biotechnol. 8:340-344 (1990); Meins and Kunz, In: GeneInactivation and Homologous Recombination in Plants, Paszkowski (ed.),pp. 335-348, Kluwer Academic, Netherlands (1994)) (Kinney, InducedMutations and Molecular Techniques for Crop Improvement, Proceedings ofa Symposium 19-23 Jun. 1995 (jointly organized by IAEA and FA)), pages101-113 (IAEA-SM 340-49).

It is understood that one or more of the nucleic acids of the inventionmay be introduced into a plant cell and transcribed using an appropriatepromoter with such transcription resulting in the cosuppression of anendogenous protein. Such nucleic acid molecules may be operably linkedto the same promoter in polycistronic configuration or to differentpromoters.

Antisense approaches are a way of preventing or reducing gene functionby targeting the genetic material (Mol et al., FEBS Lett. 268:427-430(1990)). The objective of the antisense approach is to use a sequencecomplementary to the target gene to block its expression and create amutant cell line or organism in which the level of a single chosenprotein is selectively reduced or abolished. Antisense techniques haveseveral advantages over other ‘reverse genetic’ approaches. The site ofinactivation and its developmental effect can be manipulated by thechoice of promoter for antisense genes or by the timing of externalapplication or microinjection. Antisense can manipulate its specificityby selecting either unique regions of the target gene or regions whereit shares homology to other related genes (Hiatt et al., In: GeneticEngineering, Setlow (ed.), Vol. 11, New York: Plenum 49-63 (1989)).

Antisense RNA techniques involve introduction of RNA that iscomplementary to the target mRNA into cells, which results in specificRNA:RNA duplexes being formed by base pairing between the antisensesubstrate and the target mRNA (Green et al., Annu. Rev. Biochein.55:569-597 (1986)). Under one embodiment, the process involves theintroduction and expression of an antisense gene sequence. Such asequence is one in which part or all of the normal gene sequences areplaced under a promoter in inverted orientation so that the ‘wrong’ orcomplementary strand is transcribed into a noncoding antisense RNA thathybridizes with the target mRNA and interferes with its expression(Takayama and Inouye, Crit. Rev. Biochem. Mol. Biol. 25:155-184 (1990)).An antisense vector is constructed by standard procedures and introducedinto cells by methods including but not limited to transformation,transfection, electroporation, microinjection, and infection. The typeof transformation and choice of vector will determine whether expressionis transient or stable. The promoter used for the antisense gene mayinfluence the level, timing, tissue, specificity, or inducibility of theantisense inhibition.

It has been reported that the introduction of double-stranded RNA intocells may also be used to disrupt the function of an endogenous gene.(Fire et al., Nature 391: 806-811 (1998)). Such disruption has beendemonstrated, for example, in Caenorhabditis elegans and is oftenreferred to as RNA interference, or RNAi. (Fire et al., Nature 391:806-811 (1998)). The disruption of gene expression in C. elegans bydouble-stranded RNA has been reported to induce suppression by apost-transcriptional mechanism. (Montgomery et al., Proc. Natl. Acad.Sci. 95:15502-15507 (1998)). Evidence of gene silencing bydouble-stranded RNA has also been reported for plants. (Waterhouse etal., Proc. Natl. Acad. Sci. 95: 13959-13964 (1998)).

An intron-spliced hairpin structure reportedly may also be used toeffect post-transcriptional gene suppression. (Smith et al., Nature 407:319-320 (2000)). Reports indicate that post-transcriptional genesilencing can be induced with almost 100% efficiency by the use ofintron-spliced RNA with a hairpin structure. (Smith et al., Nature 407:319-320 (2000)).

It is understood that one or more of the nucleic acids of the inventionmay be modified in order to effect RNAi or another mode ofpost-transcriptional gene suppression.

The present invention also provides for parts of the plants,particularly reproductive or storage parts. Plant parts, withoutlimitation, include seed, endosperm, ovule, pollen, roots, tubers,stems, leaves, stalks, fruit, berries, nuts, bark, pods, seeds andflowers. In a particularly preferred embodiment of the presentinvention, the plant part is a seed.

The present invention also provides a container of over 10,000, morepreferably 20,000, and even more preferably 40,000 seeds where over 10%,more preferably 25%, more preferably 50% and even more preferably 75% or90% of the seeds are seeds derived from a plant of the presentinvention.

The present invention also provides a container of over 10 kg, morepreferably 25 kg, and even more preferably 50 kg seeds where over 10%,more preferably 25%, more preferably 50% and even more preferably 75% or90% of the seeds are seeds derived from a plant of the presentinvention.

Any of the plants or parts thereof of the present invention may beprocessed to produce a feed, meal, protein, or oil preparation. Aparticularly preferred plant part for this purpose is a seed. In apreferred embodiment the feed, meal, protein or oil preparation isdesigned for livestock animals or humans, or both. Methods to producefeed, meal, protein and oil preparations are known in the art. See, forexample, U.S. Pat. Nos. 4,957,748, 5,100,679, 5,219,596, 5,936,069,6,005,076, 6,146,669, and 6,156,227. In a preferred embodiment, theprotein preparation is a high protein preparation. Such a high proteinpreparation preferably has a protein content of greater than 5% w/v,more preferably 10% w/v, and even more preferably 15% w/v. In apreferred oil preparation, the oil preparation is a high oil preparationwith an oil content derived from a plant or part thereof of the presentinvention of greater than 5% w/v, more preferably 10% w/v, and even morepreferably 15% w/v. In a preferred embodiment the oil preparation is aliquid and of a volume greater than 1, 5, 10 or 50 liters. The presentinvention provides for oil produced from plants of the present inventionor generated by a method of the present invention. Such an oil mayexhibit enhanced oxidative stability. Also, such oil may be a minor ormajor component of any resultant product. Moreover, such oil may beblended with other oils. In a preferred embodiment, the oil producedfrom plants of the present invention or generated by a method of thepresent invention constitutes greater than 0.5%, 1%, 5%, 10%, 25%, 50%,75% or 90% by volume or weight of the oil component of any product. Inanother embodiment, the oil preparation may be blended and canconstitute greater than 10%, 25%, 35%, 50% or 75% of the blend byvolume. Oil produced from a plant of the present invention can beadmixed with one or more organic solvents or petroleum distillates.

In one embodiment, an oil of the present invention has an oilcomposition that is 50% or greater oleic acid and 10% or less linolenicacid. In another embodiment, an oil of the present invention has an oilcomposition that is 60% or greater oleic acid and 7% or less linolenicacid. In another embodiment, an oil of the present invention has an oilcomposition that is 65% or greater oleic acid and 5% or less linolenicacid, preferably 4% or less linolenic acid, and more preferably 3% orless linolenic acid.

In another embodiment, an oil of the present invention has an oilcomposition that is between 50% and 90% oleic acid and 10% or lesslinolenic acid. In another embodiment, an oil of the present inventionhas an oil composition that is between 60% and 80% oleic acid and 7% orless linolenic acid. In another embodiment, an oil of the presentinvention has an oil composition that is between 65% and 75% oleic acidand 5% or less linolenic acid, preferably 4% or less linolenic acid, andmore preferably 3% or less linolenic acid.

In another embodiment, an oil of the present invention has an oilcomposition that is 80% or greater, more preferably 90% or greater oleicacid and 5% or less linolenic acid, preferably 4% or less linolenicacid, and more preferably 3% or less linolenic acid. In anotherembodiment, an oil of the present invention has an oil composition thatis 50% or greater oleic acid, more preferably 60% or greater, 70% orgreater, 80% or greater, or 90% or greater oleic acid. In anotherembodiment, an oil of the present invention has an oil composition thatis 10% or less linolenic acid, preferably 5% or less, 4% or less, or 3%or less linolenic acid.

Plants of the present invention can be part of or generated from abreeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc). Selected, non-limiting approaches, for breedingthe plants of the present invention are set forth below. A breedingprogram can be enhanced using marker assisted selection of the progenyof any cross. It is further understood that any commercial andnon-commercial cultivars can be utilized in a breeding program. Plantsof the present invention can be part of or generated from a breedingprogram. The choice of breeding method depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc). Selected, non-limiting approaches, for breeding theplants of the present invention are set forth below. A breeding programcan be enhanced using marker assisted selection of the progeny of anycross. It is further understood that any commercial and non-commercialcultivars can be utilized in a breeding program.

In a non-limiting example of breeding, a soybean FAD2-1A intronsuppressed line can be used to pollinate a low linolenic soybean FAD3mutant line derived from a spontaneous mutation, or derived from but notlimited to methods such as Targeted Induced Local Lesions in Genomes(TILLING) (McCallum et al., Nature Biotech, 18:455-457 (2000)), genereplacement via RNA/DNA chimeric oligonucleotides, homologousrecombination, T-DNA or transposan mutagenesis. RNA from soybean seedscontaining both one or more expressed FAD2 intron regions and FAD3mutations, including knock out, can be screened using Northern blots (asdescribed in Example 5) to determine the levels of FAD2-1, FAD2-2,FAD3-1A, FAD3-1C and FAD3-1B transcripts. The resulting plant isexpected to have the attributes of both parents. Soybean plants withundetectable or low levels of FAD2 or FAD3 transcripts can be screenedfor fatty acid composition.

Plants of the present invention can be part of or generated from abreeding program, for example, where a soybean FAD3-1A, FAD3-1B, and/orFAD3-1 C intron suppressed line is used to pollinate soybean plants withelevated levels of oleic acid containing a FAD2 mutant line derived froma spontaneous mutation, or derived from non-limiting methods such as,for example, TILLING, gene replacement via RNA/DNA chimericoligonucleotides, homologous recombination, T-DNA or transposanmutagenesis. RNA from soybean seeds containing both one or moreexpressed FAD3 intron regions and FAD2 mutations, including knock out,can be screened using Northern blotting (as described in Example 5) todetermine the levels of FAD2-1, FAD2-2, FAD3-1A, FAD3-1B, and FAD3-1Ctranscripts. Soybean plants with undetectable or low levels of FAD2 orFAD3 transcripts can be screened for fatty acid composition. Resultingplants are expected to have the attributes of both parents.

The development of new cultivars requires the development and selectionof varieties, the crossing of these varieties and the selection ofsuperior hybrid crosses. The hybrid seed can be produced by manualcrosses between selected male-fertile parents or by using male sterilitysystems. Hybrids are selected for certain single gene traits such as podcolor, flower color, seed yield, pubescence color, or herbicideresistance, which indicate that the seed is truly a hybrid. Additionaldata on parental lines, as well as the phenotype of the hybrid,influence the breeder's decision whether to continue with the specifichybrid cross. The parents in such hybrid production can be, for example,a soybean FAD2-1A intron suppressed line, a soybean FAD3-1A, FAD3-1Band/or FAD3-1C intron suppressed line or an intron suppressed linecontaining FAD2-1A, FAD3-1A, FAD3-1B and/or FAD3-1C in any desiredcombination. Any of these parents may be crossed with, for example, anynaturally occurring or manmade mutant line with increased levels ofoleic acid and/or decreased levels of linolenic acid.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait, such as transgenes, into a desirablehomozygous cultivar or inbred line, which is the recurrent parent. Thesource of the trait to be transferred is called the donor parent. Theresulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent. After the initial cross, individuals possessing thephenotype of the donor parent are selected and repeatedly crossed(backcrossed) to the recurrent parent. The resulting parent is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. The donor parent insuch a backcross production may be, for example, a soybean FAD2-1Aintron suppressed line or a soybean FAD3-1A, FAD3-1B and/or FAD3-1Cintron suppressed line or an intron suppressed line containing FAD2-1A,FAD3-1A, FAD3-1B and/or FAD3-1C in any desired combination. Therecurrent parent can be for example any naturally occurring or manmademutant line with increased levels of oleic acid and/or decreased levelsof linolenic acid.

Computer Readable Medium

The nucleotide sequence provided in SEQ ID NO: 1 through 15, 18, 19, 22,23, or fragment thereof or complement thereof, or a nucleotide sequenceat least 50%, 60%, or 70% identical, preferably 80%, 85% identical, orespecially preferably 90%, or 95% identical, or particularly highlypreferably 97%, 98%, or 99% identical to the sequence provided in SEQ IDNO: 1 through 15, 18, 19, 22, 23, or fragment thereof or complementthereof, can be “provided” in a variety of media to facilitate use. Sucha medium can also provide a subset thereof in a form that allows askilled artisan to examine the sequences.

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, “computer readable media” refers to any medium that can be readand accessed directly by a computer. Such media include, but are notllinited to: magnetic storage media, such as floppy disk, hard disc,storage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemedia can be used to create a manufacture comprising a computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. A skilled artisan can readily adopt any ofthe presently known methods for recording information on computerreadable media to generate media comprising the nucleotide sequenceinformation of the present invention. A variety of data storagestructures are available to a skilled artisan for creating a computerreadable medium having recorded thereon a nucleotide sequence of thepresent invention. The choice of the data storage structure willgenerally be based on the means chosen to access the stored information.In addition, a variety of data processor programs and formats can beused to store the nucleotide sequence information of the presentinvention on computer readable media. The sequence information can berepresented in a word processing text file, formatted incommercially-available software such as Word Perfect and Microsoft Word,or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. A skilled artisancan readily adapt any number of data processor structuring formats (e.g.text file or database) in order to obtain computer readable media havingrecorded thereon the nucleotide sequence information of the presentinvention.

By providing one or more nucleotide sequences of the present invention,a skilled artisan can routinely access the sequence information for avariety of purposes. Computer software is publicly available whichallows a skilled artisan to access sequence information provided in acomputer readable medium. Software which implements the BLAST (Altschulet al., J. Mol. Biol. 215: 403-410 (1990)) and BLAZE (Brutlag et al.,Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system canbe used to identify non-coding regions and other nucleic acid moleculesof the present invention within the genome that contain homology tonon-coding regions from other organisms. Such non-coding regions may beutilized to affect the expression of commercially important proteinssuch as enzymes used in amino acid biosynthesis, metabolism,transcription, translation, RNA processing, nucleic acid and proteindegradation, protein modification, and DNA replication, restriction,modification, recombination, and repair.

The present invention further provides systems, particularlycomputer-based systems, which contain the sequence information describedherein. Such systems are designed to identify commercially importantfragments of the nucleic acid molecules of the present invention. Asused herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the nucleotidesequence information of the present invention. The minimum hardwaremeans of the computer-based systems of the present invention comprises acentral processing unit (CPU), input means, output means, and datastorage means. A skilled artisan can readily appreciate that any one ofthe currently available computer-based systems is suitable for use inthe present invention.

As indicated above, the computer-based systems of the present inventioncomprise a data storage means having stored therein a nucleotidesequence of the present invention and the necessary hardware means andsoftware means for supporting and implementing a search means. As usedherein, “data storage means” refers to memory that can store nucleotidesequence information of the present invention, or a memory access meanswhich can access manufactures having recorded thereon the nucleotidesequence information of the present invention. As used herein, “searchmeans” refers to one or more programs which are implemented on thecomputer-based system to compare a target sequence or target structuralmotif with the sequence information stored within the data storagemeans. Search means are used to identify fragments or regions of thesequence of the present invention that match a particular targetsequence or target motif. A variety of known algorithms are disclosedpublicly and a variety of commercially available software for conductingsearch means are available and can be used in the computer-based systemsof the present invention. Examples of such software include, but are notlimited to, MacPattern (EMBL), BLASTIN, and BLASTIX (NCBIA). One of theavailable algorithms or implementing software packages for conductinghomology searches can be adapted for use in the present computer-basedsystems.

The most preferred sequence length of a target sequence is from about 10to 100 amino acids or from about 30 to 300 nucleotide residues. However,it is well recognized that during searches for commercially importantfragments of the nucleic acid molecules of-the present invention, suchas sequence fragments involved in gene expression and proteinprocessing, the target sequence may be of shorter length.

As used herein, “a target structural motif,” or “target motif” refers toany rationally selected sequence or combination of sequences in whichthe sequences are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif. There are avariety of target motifs known in the art. Protein target motifsinclude, but are not limited to, enzymatic active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, cis elements, hairpin structures, and inducibleexpression elements (protein binding sequences).

Thus, the present invention further provides an input means forreceiving a target sequence, a data storage means for storing the targetsequences of the present invention sequence identified using a searchmeans as described above, and an output means for outputting theidentified homologous sequences. A variety of structural formats for theinput and output means can be used to input and output information inthe computer-based systems of the present invention. A preferred formatfor an output means ranks fragments of the sequence of the presentinvention by varying degrees of homology to the target sequence ortarget motif. Such presentation provides a skilled artisan with aranking of sequences which contain various amounts of the targetsequence or target motif and identifies the degree of homology containedin the identified fragment.

A variety of comparing means can be used to compare a target sequence ortarget motif with the data storage means to identify sequence fragmentssequence of the present invention. For example, implementing softwarethat implement the BLAST and BLAZE algorithms (Altschul et al., J. Mol.Biol. 215:403-410 (1990)) can be used to identify non-coding regionswithin the nucleic acid molecules of the present invention. A skilledartisan can readily recognize that any one of the publicly availablehomology search programs can be used as the search means for thecomputer-based systems of the present invention.

The following examples are illustrative and not intended to be limitingin any way.

EXAMPLES Example 1 Cloning of Desaturase Genomic Sequences

1A. Soybean Δ12 Desaturase (FAD2-1)

A soybean FAD2-1A sequence is identified by screening a soybean genomiclibrary using a soybean FAD2-1 cDNA probe. Three putative soy FAD2-1clones are identified and plaque purified. Two of the three soy FAD2-1clones are ligated into pBluescript II KS+ (Stratagene) and sequenced.Both genomic clones (14-1 and 11-12) are the same and matched thecorresponding sequence in the soy FAD2-1 cDNA exactly. A sequence of theentire FAD2-1A clone is provided in SEQ ID NO:15.

Prior to obtaining a full length clone, a portion of the FAD2-1A genomicclone is PCR amplified using PCR primers designed from the 5′untranslated sequence (Primer 12506, 5′-ATACAA GCCACTAGGCAT-3′, SEQ IDNO:16) and within the cDNA (Primer 11698:5′-GATTGGCCATGCAATGAGGGAAAAGG-3′, SEQ ID NO:17). The resulting PCRproduct is cloned into the vector pCR 2.1 (Invitrogen, Carlsbad, Calif.)and sequenced. A soy FAD2-1A partial genomic clone (SEQ ID NO:18) withan intron region (SEQ ID NO:1) is identified by comparison to thesoybean cDNA sequence using the Pustell comparison program in Macvector.The FAD2-1A intron sequence (SEQ ID NO:1) begins after the ATG startcodon, and is 420 bases long.

A second FAD2-1 gene family member is also identified and cloned, and isreferred to herein as FAD2-1B. The soy FAD2-1B partial genomic clone(SEQ ID NO:19) has a coding region (base pairs 1783-1785 and 2191-2463)and an intron region (base pairs 1786-2190) which are identified bycomparison to the soybean cDNA sequence using the Pustell comparisonprogram in Macvector. The FAD2-1B intron sequence (SEQ ID NO:2) beginsafter the ATG start codon and is 405 bases long. Other regions in theFAD2-1B partial genomnic clone (SEQ ID NO:19) include a promoter (basepairs 1-1704) (SEQ ID NO:22) and 5′UTR (base pairs 1705-1782).

1B. Soybean A15 Desaturase (FAD3)

A partial soybean FAD3-1A genomic sequence is PCR amplified from soybeanDNA using primers 10632, 5′-CUACUACUACUACTCGAGACAAAGCCTTTAGCCTATG-3′(SEQ ID NO:20), and 10633: 5′-CAUCAUCAUCAUGGATCCCATGTCTCTCTATGCAAG-3′(SEQ ID NO:21). The Expand Long Template PCR system (Roche AppliedSciences, Indianapolis) is used according to the manufacturer'sdirections. The resulting PCR products are cloned into the vector pCR2.1 (Invitrogen) and sequenced. A soy FAD3-1A partial genomic clonesequence (SEQ ID NO: 23) and intron regions are confirmed by comparisonsto the soybean FAD3-1A cDNA sequence using the Pustell program inMacvector.

From the identified partial genomic soybean FAD3-1A sequence (SEQ IDNO:23), seven introns are identified: FAD3-1A intron #1(SEQ ID NO:5),FAD3-1A intron #2 (SEQ ID NO:6), FAD3-1A intron #3A (SEQ ID NO:7),FAD3-1A intron #4 (SEQ ID NO:8), FAD3-1A intron #5 (SEQ ID NO:9),FAD3-1A intron #3B (SEQ ID NO:10), and FAD3-1A intron #3C (SEQ IDNO:11). FAD3-1A intron #1 is 191 base pairs long and is located betweenpositions 294 and 484, FAD3-1A intron #2 is 346 base pairs long and islocated between positions 577 and 922, FAD3-1A intron #3A is 142 basepairs long and is located between positions 991 and 1132, FAD3-1A intron#3B is 98 base pairs long and is located between positions 1224 and1321, FAD3-1A intron #3C is 115 base pairs long and is located betweenpositions 1509 and 1623, FAD3-1A intron #4 is 1228 base pairs long andis located between positions 1707 and 2934, and FAD3-1A intron #5 is 625base pairs long and is located between positions 3075 and 3699.

A partial soybean FAD3-1B genomic sequence is PCR amplified from soybeanDNA using primers 19386, 5′-GGTAACAGAGAAAGAAACATTTGAGC-3′ and 19369:5′-GCATGCTAACAAAAGTAAGTGC-3′. The Expand Long Template PCR system (RocheApplied Sciences, Indianapolis) is used according to the manufacturer'sdirections. The resulting PCR products are cloned into the vector pCR2.1 TOPO (Invitrogen) and sequenced. A soy FAD3-1B partial genomic clonesequence and intron regions are confirmed by comparisons to the soybeanFAD3-1B cDNA sequence using the Pustell program in Sequencher. From theidentified partial genomic soybean FAD3-1B sequence, seven introns areidentified: FAD3-1B intron #1, FAD3-1B intron #2, FAD3-1B intron #3A,FAD3-1B intron #4 (SEQ ID NO:13), FAD3-1B intron #5, FAD3-1B intron #3B,and FAD3-1B intron #3C (SEQ ID NO:12).

Example 2 Expression Constructs

2A. Construction of pCGN5468, pCGN5469, pCGN5471, pCGN5485, and pCGN5486

The FAD2-1A intron sequence (SEQ ID NO: 1) is amplified via PCR usingthe FAD2-1A partial genomic clone (SEQ ID NO:18) as a template andprimers 12701 (5′-ACGAATTCCTCGAGGTAAA TTAAATTGTGCCTGC-3′ (SEQ ID NO:24))and 12702 (5′-GCGAGATCTATCG ATCTGTGTCAAAGTATAAAC-3′ (SEQ ID NO:25)). Theresulting amplification products are cloned into the vector pCR 2.1(Invitrogen) and sequenced. The FAD2-1A intron (SEQ ID NO: 1) is thencloned into the expression cassette, pCGN3892, in sense and antisenseorientations. The vector pCGN3892 contains the soybean 7S promoter and apea RBCS 3′. Both gene fusions are then separately ligated intopCGN9372, a vector that contains the CP4 gene regulated by the FMVpromoter. The resulting expression constructs (pCGN5469 sense (FIG. 2)and pCGN5471 antisense (FIG. 3)) are used for transformation of soybeanusing biolistic methods described below.

The FAD2-1B intron sequence (SEQ ID NO: 2) is amplified via PCR usingthe FAD2-1B partial genomic clone (SEQ ID NO: 19) as a template andprimers 13883 (5′-GCGATCGATGTATGATGCTAAATTAAATTGTGCCTG-3′ (SEQ IDNO:28)) and 13876 (5′-GCGGAATTCCTGTGTCAAAGTATAAAGAAG-3′ (SEQ ID NO:29)).The resulting amplification products are cloned into the vector pCR 2.1(Invitrogen) and sequenced. The FAD2-1B intron is fused to the 3′ end ofthe FAD2-1A intron in plasmids pCGN5468 (FIG. 1) (containing the soybean7S promoter fused to the FAD2-1A intron (sense) and a pea RBCS 3′) orpCGN5470 (containing the soybean 7S promoter fused to the FAD2-1A intron(antisense) and a pea RBCS 3′) in sense or antisense orientation,respectively. The resulting intron combination fusions are then ligatedseparately into pCGN9372, a vector that contains the CP4 gene regulatedby the FMV promoter. The resulting expression constructs (pCGN5485 (FIG.4), FAD2-1A & FAD2-1B intron sense; and pCGN5486 (FIG. 5), FAD2-1A &FAD2-1B intron antisense) are used for transformation of soybean usingbiolistic methods described below.

2B. PCR Amplification of FAD3-1A Introns

Four of the seven introns identified from the soybean FAD3-1A genomicclone are PCR amplified using the FAD3-1A partial genomic clone astemplate and primers as follows: FAD3-1A intron #1, primers 12568:5′-GATCGATGCCCGGGGTAATAATTTTTGTGT-3′ (SEQ ID NO:30) and 12569:5′-CACGCCTCGAGTGTTCAATTCAATCAATG-3′ (SEQ ID NO:31); FAD3-1A intron #2,primers 12514: 5′-CACTCGAGTTAGTTCATACTGGCT-3′ (SEQ ID NO:32) and 12515:5′-CGCATCGATTGCAAAATCCATCAAA-3′ (SEQ ID NO:33); FAD3-1A intron #4,primers 10926: 5′-CUACUACUACUACTCGAGCGTAAATAGTGGGTGAACAC-3′ (SEQ IDNO:34) and 10927: 5′-CAUCAUCAUCAUCTCGAGGAATTCGTCCATTTTAGTACACC-3′ (SEQID NO:35); FAD3-1A intron #5, primers 10928: 5′-CUACUACUACUACTCGAGGCGCGTACATTTTATTGCTTA-3′ (SEQ ID NO:36) and 10929: 5′-CAUCAUCAUCAUCTCGAGGAATTCTGCAGTGAATCCAAATG-3′ (SEQ ID NO:37). The resulting PCRproducts for each intron are cloned into the vector pCR 2.1 (Invitrogen)and sequenced.

2C. Construction of pCGN5455, pCGN5459, pCGN5456, pCGN5460, pCGNS466,and pCGN5473

FAD3-1A introns #1, #2, #4 and #5 are each ligated separately intopCGN3892, in sense or antisense orientations. pCGN3892 contains thesoybean 7S promoter and a pea RBCS 3′. These fusions are ligated intopCGN9372, a vector that contains the CP4 gene regulated by the FMVpromoter for transformation into soybean. The resulting expressionconstructs (pCGN5455 (FIG. 12), FAD3-1A intron #4 sense; pCGN5459 (FIG.13), containing FAD3-1A intron #4 antisense; pCGN5456, FAD3 intron #5sense; pCGN5460, FAD3-1A intron #5 antisense; pCGN5466 (FIG. 7),containing FAD3-1A intron #2 antisense; pCGN5473 (FIG. 9), containingFAD3-1A intron #1 antisense) are used for transformation of soybeanusing biolistic methods described below.

Introns #3C and #4 are also PCR amplified from a second FAD3 gene familymember (FAD3-1B). Soybean FAD3-1B introns #3C and #4 are PCR amplifiedfrom soybean DNA using the following primers, 5′ CATGCTTTCTGTGCTTCTC 3′(SEQ ID NO:26) and 5′ GTTGATCCAACCATAGTCG 3′ (SEQ ID NO:27). The PCRproducts are cloned into the vector pCR 2.1 (Invitrogen) and sequenced.Sequences for FAD3-1B introns #3C and #4 are provided in SEQ ID NOs:12and 13, respectively.

Intron #4 from three soybean FAD3 gene family members, FAD3-1A, FAD3-1Band FAD3-1C is PCR amplified. Intron #4 from the FAD3-1A gene is PCRamplified using the FAD3-1A partial genomic clone as template andprimers primers 10926: 5′-CUACUACUACUACTCGAGCGTAAATAGTGGGTGAACAC-3′ (SEQID NO:34) and 10927: 5′-CAUCAUCAUCAUCTCGAGGAATTCGTCCATTTTAGTACACC-3′(SEQ ID NO:35). Intron #4 from the FAD3-1B gene is PCR amplified usingthe soybean genomic DNA as template and primers #17823: GTATCCCATTTAACACand #17824: CTGTGAAATTACATATAG. Intron #4 from the FAD3-1C gene is PCRamplified using the soybean genomic DNA as template and primers #17826:GCGCCGCTCGAGCTGTCCATTTTTGTACAC and #17825:CCGGCGCTCGAGGTAACAAAAATAAATAGAAAATAGTGAGTG. The resulting PCR productsfor each intron were cloned into the vector pCR 2.1 (Invitrogen) andsequenced. FAD3-1A intron, #4 was ligated into the pCGN3892 in senseorientation. The resulting expression cassette, pCGN5453, contained the7S alpha′ promoter fused to the FAD3-1A intron #4, with a pea RBCS 3′.

2D. Construction of pMON68521

To construct pMON68521 (FIG. 10), pCGN5453 (7S alpha′ promoter fused tothe FAD3-1 A intron #4, with a pea RBCS 3′) and KWHIT032858 (PCR2.1containing the FAD3-1B intron #4) are digested with EcoRI and ligatedtogether to form KWHIT03004 (7Salpha′ promoter fused to the FAD3-1Aintron #4 and FAD3-1B intron #4, with a pea RBCS 3′). KWHIT03004 isdigested with XhoI and KAWHIT032980 (FAD3-1C intron #4 in PCR2.1) isdigested with EcoRI and the ends of both digested plasmids are filledand subsequently ligated. The resulting plasmid is KAWHIT030005(7Salpha′ promoter fused to the FAD3-1A intron #4, FAD3-1B intron #4,and FAD3-1B intron #4, with a pea RBCS 3′). KAWHIT030005 is digestedwith SacI and the ends are filled using the Klenow fragment of T4polymerase to create blunt ends. pCGN5468 (FIG. 1, containing 7S alpha′promoter fused to the FAD2-1A intron, with a pea RBCS 3′) is digestedwith EcoRI and the ends are filled with Klenow fragment of T4 polymeraseto form blunt ends. The blunt ends of plasmids KAWHIT030005 and pCGN5468are ligated together to form KWHIT03001 (7S alpha′ promoter fused to theFAD2-1A intron, the FAD3-1A intron #4, FAD3-1B intron #4, FAD3-1C intron#4, with a pea RBCS 3′). KWHIT03001 and pMON 70276 (FMV-EF-1/CP4) areboth digested with NotI, ligated together to form pMON68521 (FIG. 10, 7Salpha′ promoter fused to the FAD2-1A intron, the FAD3-1A intron #4,FAD3-1B intron #4, FAD3-1C intron #4, with a pea RBCS 3′ andFMV-EF-1/CP4, with a pea RBCS 3′). pMON68521 was transformed into theABI strain of Agrobacterium tumefaciens and cocultivated into soybean.

2E. Construction of pMON68519

In constructing the plasmid pMON68519 (FIG. 11), pCGN5453 (containing7Salpha′ promoter fused to the FAD3-1A intron #4, with a pea RBCS 3′)and KWHIT 032858 (pCR2.1 containing the FAD1B intron #4) are digestedwith EcoRI and ligated together to form KWHIT03004 (containing 7Salpha′promoter fused to the FAD3-1A intron #4 and FAD3-1B intron #4, with apea RBCS 3′). KWHIT03004 is digested with XhoI and KAWHIT032980(containing FAD3-1C intron #4 in PCR2.1) is digested with EcoRI. Theends of both digested plasmids are filled and the plasmids aresubsequently ligated. The resulting plasmid is named KAWHIT030005(containing 7Salpha′ promoter fused to the FAD3-1A intron #4, FAD3-1Bintron #4, and FAD3-1B intron #4, with a pea RBCS 3′). KAWHIT030005 isdigested with SacI and then the DNA is treated with the Klenow fragmentof T4 polymerase to create blunt ends. pCGN7770 (containing the Napinpromoter and Napin 3′ ) is digested with XhoI and blunt-ended withKienow fragment of T4 polymerase. The blunt ends of plasmidsKAWHIT030005 and pCGN7770 are ligated together to form KWHIT03007(containing a Napin promoter fused to the FAD2-1A intron, the FAD3-1Aintron #4, FAD3-1B intron #4, FAD3-1C intron #4, with a Napin 3′).

KWHIT03007 is then digested with NotI and the ends are filled with theKlenow fragment of T4 polymerase, forming blunt ends. The Napin promoterfused to the FAD2-1 A intron, the FAD3-1A intron #4, FAD3-1B intron #4,FAD3-1C intron #4, with a Napin3′ (KWHIT03007) is blunt end ligated intopMON68504 which is digested with EcoRV. pMON68504 contains the SoyFAD2-1A intron fused to the 7s Alpha′ promoter within the 2 tDNA vectorpMON41162, which contains the CP4 gene regulated by the FMV promoter,with the pea RBCS 3′. The blunt end ligation between KWHIT03007 andpMON6850 results in the plasmid, pMON68519. pMON68519 is transformedinto the ABI strain of Agrobacterium tumefaciens and cocultivated intosoybean.

Example 3 Plant Transformation and Analysis

Linear DNA fragments containing the expression constructs for sense andantisense suppression of the Δ12 and Δ15 desaturase genes are stablyintroduced into soybean (Asgrow variety A3244 or A4922A32) by theparticle bombardment method of McCabe et al. (1988), Bio/Technology,6:923-926 or via cocultivation with Agrobacterium tumefaciens, strainABI. (Martinell, U.S. Pat. No. 6,384,310). Transformed soybean plantsare identified by selection on media containing glyphosate.

Fatty acid compositions are analyzed from seed of soybean linestransformed with the intron expression constructs using gaschromatography. R1 pooled seed and R1 single seed oil compositionsdemonstrate that the mono- and polyunsaturated fatty acid compositionswere altered in the oil of seeds from transgenic soybean lines ascompared to that of the seed from non-transformed soybean. Tables I, II,and III provide summaries of results which were obtained using thedescribed constructs. These data show that sense and antisenseexpression of non-coding regions of a desaturase gene result in themodification of the fatty acid compositions. The data also shows thatintrons can be used to obtain a variety of lines with varying fatty acidcompositions. Selections can be made from such lines depending on thedesired relative -fatty acid composition. Because each intron is able tomodify the levels of each fatty acid to varying extents, it iscontemplated that combinations of introns can be used depending on thedesired compositions.

TABLE I Orientation Event Oleic Linoleic Linolenic FAD2 wildtype(control) 5469-5 null R1 pool 18.15% 55.59% 7.97% 10 seed average 13.89%55.89% 9.067%  5469-27 null R1 pool 19.15% 54.62% 9.32% A4922 15.75% 56.1% 8.75% 5471-13 null R1 pool 17.02% 56.49% 9.08% 10 seed average13.86% 56.14% 9.49% A4922 14.95% 55.95% 9.07% full length cDNA sense5462-133 R1 pool   84%  2.17% 1.55% (control) best 5462-133 R1 seed  84%  0.59% 1.76% intron 1 sense 5469-6 R1 pool 29.93% 46.53% 5469-8 R1pool  36.5% 42.11% 5.98% best 5469-6 R1 seed 44.41% 29.34% 6.68% best5469-8 R1 seed 41.26% 33.16% 5.74% 5469-14 R1 pool 61.06% 16.42% 7.75%5469-20 R1 pool 48.89% 31.61% 4.89% 5469-22 R1 pool   80%  2.97% 4.78%best 5469-14 R1 seed 62.21% 11.97% 8.81% 5485-3 R1 pool 63.54% 14.09%7.32% 5485-53 R1 pool 47.58% 27.64% 7.81% antisense 5471-8 R1 pool31.05% 43.62% 7.07% 5471-2 R1 pool 27.98% 48.88% 6.83% 5471-26 R1 pool32.66% 44.54% 6.76% best 5471-8 R1 seed  57.4% 23.37% 5.73% best 5471-2R1 seed 28.08% 46.14% 6.52% best 5471-26 R1 seed  43.3% 34.15%  5.6%5486-33 R1 pool 32.37% 43.66% 6.87% 5486-12 R1 pool 27.32% 46.97%  6.4%5486-40 R1 pool 26.79% 48.72% 6.55% FAD3 wildtype (control) 5473-7 nullR1 pool 15.65% 56.74% 9.55% A4922 R1 pool 19.84% 56.79% 7.48% fulllength cDNA sense 5464-50 R1 pool 18.06% 62.03% 2.75% (control) best5464-50 R1 seed 17.08% 62.44% 1.72% intron 1 antisense 5473-8 R1 pool33.47% 45.97% 5.54% 5473-1 R1 pool 33.34% 42.67% 7.59% intron 2antisense 5466-20 R1 pool 28.43% 48.83% 6.37% 5466-16 R1 pool 27.61%49.92% 5.96% intron 4 sense 5455-19 R1 pool 40.35% 39.97% 4.61% 5455-10R1 pool 35.14% 43.59% 5.53% 5455-57 R1 pool 38.04% 42.44% 5.24% 5455-76R1 pool 37.24% 42.42% 5.37% 5455-107 R1 pool 36.44% 42.72% 5.62% best5455-57 R1 seed 45.36% 35.55% 4.92% best 5455-76 R1 seed  35.3% 43.54%5.53% best 5455-107 R1 seed 45.56% 34.85% 5.12% antisense 5459-2 R1 pool 34.5% 43.87% 5.59% 5459-6 R1 pool 33.78% 44.12% 5.62% 5459-20 R1 pool28.26% 49.48%  5.5% best 5459-2 R1 seed 61.45% 23.45% 3.38% best 5459-6R1 seed 53.51% 29.68% 3.53% best 5459-20 R1 seed   30% 50.55% 4.15%intron 5 sense 5456-38 R1 pool 28.23% 49.59% 6.74% 5456-62 R1 pool28.94% 48.66% 6.25% best 5456-62 R1 seed  29.5% 43.69%  5.4% antisense5460-9 R1 pool 29.78% 48.57% 5.54% 5460-21 R1 pool 28.37% 49.79% 5.54%best 5460-21 R1 seed 35.18% 40.52% 5.33%

TABLE II Oil Composition data for seeds containing pMON68521 R1 singleseed data Construct Strain ID 16:0 18:0 18:1 18:2 18:3 PMON68521GM_A32162 12.0 3.4 42.8 35.5 5.3 PMON68521 GM_A32162 11.5 2.6 39.4 40.05.2 PMON68521 GM_A31619 10.4 2.8 39.1 40.4 5.8 PMON68521 GM_A32162 11.92.6 36.7 41.9 5.8 PMON68521 GM_A32162 12.2 2.5 34.9 43.0 6.3 PMON68521GM_A32162 13.0 2.8 30.4 46.6 6.0 PMON68521 GM_A31610 12.4 1.9 28.3 49.87.1 PMON68521 GM_A32162 11.9 2.9 26.5 51.6 6.1 PMON68521 GM_A31792 13.23.3 25.3 50.2 7.2 PMON68521 GM_A31395 12.5 3.7 25.1 50.2 6.7 PMON68521GM_A31393 13.1 3.5 24.1 51.9 5.6 PMON68521 GM_A31615 14.0 2.2 24.0 52.56.7 PMON68521 GM_A32209 12.5 3.5 23.7 51.5 7.6 PMON68521 GM_A31612 11.83.0 23.7 51.1 9.5 PMON68521 GM_A32209 12.6 3.3 23.6 52.2 7.2 PMON68521GM_A32209 12.4 3.2 23.0 53.1 7.2 PMON68521 GM_A31489 12.3 3.2 22.5 54.06.9 PMON68521 GM_A32252 12.7 4.4 22.3 52.4 7.1 PMON68521 GM_A32162 12.63.1 22.2 54.9 6.2 PMON68521 GM_A32089 13.5 3.2 22.2 52.4 7.9 PMON68521GM_A31393 12.6 4.3 22.2 53.0 5.7 PMON68521 GM_A31610 12.5 2.7 21.8 55.56.9 PMON68521 GM_A31610 12.6 2.4 21.7 55.8 6.9 PMON68521 GM_A31656 12.73.6 21.6 53.2 8.0 PMON68521 GM_A31612 12.3 3.6 21.6 54.0 7.2 PMON68521GM_A31610 13.2 2.6 21.0 56.2 6.5 PMON68521 GM_A31604 13.5 3.1 20.4 55.37.0 PMON68521 GM_A31610 13.2 2.6 20.4 56.7 6.4 PMON68521 GM_A31489 12.83.1 20.1 55.5 7.1 PMON68521 GM_A31525 12.2 3.0 20.1 57.2 6.3 A3244 13.94.1 15.8 56.3 9.0 A3244 13.7 4.1 14.2 57.6 9.3 A3244 13.6 4.3 14.1 57.49.7 A3244 13.9 4.1 14.1 56.9 10.0 A3244 13.8 4.4 13.6 57.6 9.8 A324414.2 4.8 13.6 56.8 9.5 A3244 14.2 4.3 13.2 56.5 10.8 A3244 14.0 4.2 13.157.0 10.6 A3244 14.0 4.5 12.9 57.3 10.3

TABLE III Oil Composition data for seeds containing pMON68519 R1 singleseed data Construct Strain ID 16:0 18:0 18:1 18:2 18:3 PMON68519GM_A29911 13.0 4.8 40.7 34.4 5.2 PMON68519 GM_A29911 12.3 3.8 38.8 37.55.8 PMON68519 GM_A29911 12.3 3.3 34.0 42.6 6.5 PMON68519 GM_A32856 12.93.5 33.6 42.2 6.5 PMON68519 GM_A32856 12.6 3.1 33.3 43.4 6.4 PMON68519GM_A32856 13.0 3.1 31.3 45.4 6.1 PMON68519 GM_A32856 12.7 3.2 28.9 47.36.6 PMON68519 GM_A29911 12.9 4.0 28.7 46.5 6.7 PMON68519 GM_A29911 12.23.1 28.1 47.4 6.8 PMON68519 GM_A32856 13.3 3.2 26.5 48.7 7.1 PMON68519GM_A32856 13.2 3.2 26.5 49.3 6.8 PMON68519 GM_A29911 13.1 3.2 26.1 49.47.1 PMON68519 GM_A32856 13.2 3.6 25.9 48.9 7.1 PMON68519 GM_A32857 13.33.1 23.3 52.2 7.4 PMON68519 GM_A29911 12.8 3.5 23.3 52.5 6.6 PMON68519GM_A29911 12.6 3.1 21.4 51.9 9.0 PMON68519 GM_A29911 13.4 3.6 20.1 53.67.9 A3244 13.2 4.3 16.5 55.7 9.7 A3244 13.5 3.2 16.3 56.8 9.2 A3244 13.73.4 15.4 57.7 9.2 A3244 13.9 3.3 15.1 57.9 9.1 A3244 13.8 3.6 14.3 58.39.2 A3244 13.5 3.4 13.6 58.2 10.4 A3244 13.5 3.9 12.5 58.4 11.2 A324414.6 3.8 12.1 58.2 10.8 A3244 14.5 3.9 11.8 57.8 11.4

Example 4

Linear DNA fragments containing dsRNAi constructs for suppression of theΔ12 and Δ15 desaturase genes are stably introduced into soybean (Asgrowvariety A3244 or A4922A32) via cocultivation with Agrobacteriumtumefaciens, strain ABI. (Martinell, U.S. Pat. No. 6,384,301) or by theparticle bombardment method of McCabe et al. (1988), Bio/Technology,6:923-926. Introduced constructs include: (1) 7S promoter-FAD2-1A senseintron-FAD3-1A sense intron-FAD3-1B sense intron-spliceable FAD3 intron#5-FAD3-1B antisense intron-FAD3-1A antisense intron-FAD2-1A antisenseintron-pea rbcS; (2) 7S promoter-FAD3-1A sense intron-FAD3-1B senseintron-spliceable FAD3 intron #5-FAD3-1B antisense intron-FAD3-1Aantisense intron-pea rbcS; (3) 7S promoter-FAD2-1A sense intron-FAD3-1Asense intron-spliceable FAD3 intron #5-FAD3-1A antisense intron-FAD2-1Aantisense intron-pea rbcS. Representative sequences for FAD2-1A,FAD2-1B, FAD2-2B, FAD3-1A, FAD3-1B, and FAD3-1C introns may be found,without limitation, in U.S. application Ser. No. 10/176,149, filed Jun.21, 2002, and U.S. application Ser. No. 09/638,508, filed Aug. 11, 2000,and U.S. Provisional Application Ser. No. 60/151,224, filed Aug. 26,1999, and U.S. Provisional Application Ser. No. 60/172,128, filed Dec.17, 1999. Transformed soybean plants are identified by selection onmedia containing glyphosate.

Fatty acid compositions are analyzed from transformed soybean seedscontaining iron RNAi suppression constructs. Particular lines areselected depending on the desired relative fatty acid composition.

Example 5

5A.

RNA is isolated from homozygous R2 seed from two FAD2-1 intronsuppressed lines (5469-14 and 5469-22), from two FAD2-1 cDNA suppressedlines (positive controls) (5462-87 and 5462-133), and from negativecontrols (wild type seed and seed from null segregants from each intronsuppressed event). Northern gels containing these RNA samples are probedwith the FAD2-1 cDNA. FAD2-1A transcript levels are significantlyreduced in both the intron suppressed lines and the cDNA suppressedlines relative to the negative controls. The same Northern blot isprobed with the constitutive FAD2-2 cDNA and no significant differencesin the FAD2-2 transcript levels are observed between the FAD2-1 intronsuppressed lines and the controls. In contrast, the FAD2-2 transcript inthe cDNA suppressed lines, is significantly reduced. This Northern dataindicates that the FAD2-1A intron is specifically inhibiting theaccumulation of the FAD2-1 transcript but not the FAD2-2 transcript. Apartial FAD2-2 genomic clone (SEQ ID NO: 3) is PCR amplified andsequence analysis reveals a 4.7 KB intron in the 5′ untranslated regionof the gene. The sequence of the FAD2-2 intron (SEQ ID NO: 4) shares nohomology with the FAD2-1 intron.

5B.

RNA is isolated from homozygous R2 seed from four FAD3-1A intron #4suppressed lines, from three FAD3-1B intron #4 suppressed lines, fromnegative control seed (non transformed wild type seed) and from seedfrom null segregants from each intron suppressed event). Northern gelscontaining these RNA samples are probed with the FAD3-1A 3′UTR region.Endogenous FAD3-1A transcript levels are significantly reduced in theFAD3-1A intron #4 suppressed lines relative to the wildtype or nullcontrols. The same Northern blot is probed with the FAD3-1B 3′ UTRregion and no significant differences in the endogenous FAD3-1Btranscript levels are observed relative to the FAD3-1A intron #4suppressed lines, the wildtype or null controls. The sequence of theFAD3-1A intron #4 (SEQ ID NO: 8) shares no homology with the FAD3-1Bintron #4 (SEQ ID NO: 13)

Example 6

Southern blot data indicate that there are at least two FAD3 gene familymembers. To determine the sequence of the other FAD3 gene family memberand to determine if other members exist, a FAD3-1A gene sequence is usedfor a query Blast search against the Monsanto soybean DNA sequencesdatabase. Candidate ESTs from different FAD3 gene family members areused to design primers. Using this strategy, 2 primer sets are designedbased on putative FAD3 sequences. Intron #4 regions from two other FAD3gene family members are isolated. Primers are designed from the211565_(—)1.r1040 EST (designated FAD3-1B), (5′ primer #15024:5′-CATGCTTTCTGTGCTTCTC-3′ (SEQ ID NO:26) and 3′ primer #15027:5′-GTTGATCCAACCATAGTCG-3′ (SEQ ID NO:27)) in the region corresponding tothe position of intron #4 of the FAD3-1A gene. These primers are used toPCR amplify the FAD3-1B intron #4 (SEQ ID NO:13), which when sequencedshared no sequence homology with the FAD3-1A intron #4 (SEQ ID NO: 8).The FAD3-1B gene also contains an intron #3C (SEQ ID NO: 12), which alsodid not share any homology with the FAD3-1A intron #3C (SEQ ID NO:11).

Another additional intron #4 is PCR amplified from a second EST,gsv701051989.H1 (designated FAD3-1C) using the following set of primers:5′ primer #16241: 5′-CACCATGGTCATCATCAGAAAC (SEQ ID NO:38) and the 3′primer #16242: TCACGATCCACAGTTGTGAGAC (SEQ ID NO:39). The FAD3-1C intron#4 (SEQ ID NO:14) shares 50% homology with the FAD3-1A intron #4 (SEQ IDNO: 8) and shares no homology with the FAD3-1B intron #4 (SEQ ID NO:13). The FAD3-1C EST, like the FAD3-1B EST, also contains an intron #4splice site in the same region of the gene.

Example 7 FAD2-1A/FAD3-1A Transformed Plants

7A.

A soybean FAD2-1A intron suppressed line is used to pollinate a soybeanFAD3-1A intron suppressed line that is generated according to themethodology set forth in Example 3. RNA from soybean seeds containingboth an expressed FAD2-1A intron region and FAD3-1A intron region isscreened using Northern blotting (as described in Example 5) todetermine the levels of FAD2-1, FAD2-2, FAD3-1A and FAD3-1B transcripts.Soybean plants with undetectable or low levels of FAD2-1 and FAD3-1Atranscripts are screened for fatty acid composition as set forth inExample 3.

7B.

A soybean FAD2-1A intron suppressed line is also used to pollinate a lowlinolenic soybean FAD3 mutant line derived from a mutation. RNA fromsoybean seeds containing both one ore more expressed FAD2 intron regionsand FAD3 mutations, including knock out, are screened using Northernblots (as described in Example 5) to determine the levels of FAD2-1,FAD2-2, FAD3-1A, FAD3-1C and FAD3-1B transcripts. Soybean plants withundetectable or low levels of FAD2 and FAD3 transcripts are screened forfatty acid composition as set forth in Example 3.

7C.

A soybean line with FAD3-1A, FAD3-1B, and FAD3-1C intron suppression isused to pollinate soybean plants with elevated levels of oleic acidcontaining a FAD2 mutant line derived from a spontaneous mutation.Soybean lines with FAD3-1A and FAD3-1B intron suppression, lines withFAD3-1A and FAD3-1C intron suppression, lines with FAD3-1B and FAD3-1Cintron suppression, lines with FAD3-1A intron suppression, lines withFAD3-1B intron suppression and lines with FAD3-1C intron suppression areeach used individually to pollinate soybean plants with elevated levelsof oleic acid containing a FAD2 mutant line derived from a spontaneousmutation. RNA from soybean seeds containing both one or more expressedFAD3 intron regions and FAD2 mutations, including knock out, arescreened using Northern blots as described in Example 5 to determine thelevels of FAD2-1, FAD2-2, FAD3-1A, FAD3-1B and FAD3-1C transcripts.Soybean plants with undetectable or low levels of FAD2 or FAD3transcripts are screened for fatty acid composition as set forth inExample 3.

Example 8 Single FAD2/FAD3 Constructs

Linear DNA fragments containing sense and antisense FAD2 and FAD3introns, as well as FAD2 and FAD3 introns capable of producing a dsRNA,are constructed as set forth in Table IV.

TABLE IV Structural Nucleic Structural Nucleic Acid 1 Acid 2 (sense,antisense, (sense, antisense, Construct No. Promoter 1 dsRNA) Promoter 2dsRNA) 1 CaMV FAD2-1A intron 1 CaMV FAD3-1A intron 1 2 CaMV FAD2-1Bintron 1 CaMV FAD3-1A intron 1 3 CaMV FAD2-1A intron 1 CaMV FAD3-1Aintron 4 4 CaMV FAD2-1B intron 1 CaMV FAD3-1A intron 4 5 CaMV FAD2-1Aintron 1 CaMV FAD3-1B intron 4 6 CaMV FAD2-1B intron 1 CaMV FAD3-1Bintron 4 7 CaMV FAD2-1A intron 1 CaMV FAD3-1C intron 4 8 CaMV FAD2-1Bintron 1 CaMV FAD3-1C intron 4 9 CaMV FAD2-1A intron 1 CaMV FAD2-2Bintron 1 10 CaMV FAD2-1B intron 1 CaMV FAD2-2B intron 1 11 napin FAD2-1Aintron 1 napin FAD3-1A intron 1 12 napin FAD2-1B intron 1 napin FAD3-1Aintron 1 13 napin FAD2-1A intron 1 napin FAD3-1A intron 4 14 napinFAD2-1B intron 1 napin FAD3-1A intron 4 15 napin FAD2-1A intron 1 napinFAD3-1B intron 4 16 napin FAD2-1B intron 1 napin FAD3-1B intron 4 17napin FAD2-1A intron 1 napin FAD3-1C intron 4 18 napin FAD2-1B intron 1napin FAD3-1C intron 4 19 napin FAD2-1A intron 1 napin FAD2-2B intron 120 napin FAD2-1B intron 1 napin FAD2-2B intron 1 21 7S FAD2-1A intron 1CaMV FAD3-1A intron 1 22 7S FAD2-1B intron 1 CaMV FAD3-1A intron 1 23 7SFAD2-1A intron 1 CaMV FAD3-1A intron 4 24 7S FAD2-1B intron 1 CaMVFAD3-1A intron 4 25 7S FAD2-1A intron 1 CaMV FAD3-1B intron 4 26 7SFAD2-1B intron 1 CaMV FAD3-1B intron 4 27 7S FAD2-1A intron 1 CaMVFAD3-1C intron 4 28 7S FAD2-1B intron 1 CaMV FAD3-1C intron 4 29 7SFAD2-1A intron 1 CaMV FAD2-2B intron 1 30 7S FAD2-1B intron 1 CaMVFAD2-2B intron 1 31 CaMV FAD2-1A intron 1 7S FAD3-1A intron 1 32 CaMVFAD2-1B intron 1 7S FAD3-1A intron 1 33 CaMV FAD2-1A intron 1 7S FAD3-1Aintron 4 34 CaMV FAD2-1B intron 1 7S FAD3-1A intron 4 35 CaMV FAD2-1Aintron 1 7S FAD3-1B intron 4 36 CaMV FAD2-1B intron 1 7S FAD3-1B intron4 37 CaMV FAD2-1A intron 1 7S FAD3-1C intron 4 38 CaMV FAD2-1B intron 17S FAD3-1C intron 4 39 CaMV FAD2-1A intron 1 7S FAD2-2B intron 1 40 CaMVFAD2-1B intron 1 7S FAD2-2B intron 1

As shown, each construct listed in the table can have severalconfigurations depending on the nature and orientation of the structuralnucleic acids in the construct. For example, construct 30 may beconfigured as follows: (1) 7S promoter-FAD2-1B intron 1 (sense)-CaMV-promoter-FAD2-2B intron 1 (sense); (2) 7S promoter-FAD2-1B intron 1(sense)-CaMV promoter-FAD2-2B intron 1 (antisense); (3) 7Spromoter-FAD2-1B intron 1 (sense)-CaMV promoter-FAD2-2B intron 1(dsRNA); (4) 7S promoter-FAD2-1B intron 1 (antisense)-CaMVpromoter-FAD2-2B intron 1 (sense); (5) 7S promoter-FAD2-1B intron 1(antisense)-CaMV promoter-FAD2-2B intron 1 (antisense); (6) 7Spromoter-FAD2-1B intron 1 (antisense)-CaMV promoter-FAD2-2B intron 1(dsRNA); (7) 7S promoter-FAD2-1B intron 1 (dsRNA)-CaMV promoter-FAD2-2Bintron 1 (sense); (8) 7S promoter-FAD2-1B intron 1 (dsRNA)-CaMVpromoter-FAD2-2B intron 1 (antisense); or (9) 7S promoter-FAD2-1B intron1 (dsRNA)-CaMV promoter-FAD2-2B intron 1 (dsRNA).

These constructs can be stably introduced into soybean (for example,Asgrow variety A4922 or Asgrow variety A3244) by the methods describedearlier, including the particle bombardment method of McCabe et al.(1988), Bio/Technology, 6:923-926 or Agrobacterium-mediatedtransformation (Martinell, U.S. Pat. No. 6,384,301). Transformed soybeanplants are identified by selection on media containing glyphosate. Fattyacid compositions are analyzed from seed of soybean lines transformedwith the constructs using gas chromatography.

Example 9

Linear DNA fragments containing expression constructs for sense andantisense expression of the FAD2-1 and FAD2-2 introns are stablyintroduced into soybean (for example, Asgrow variety A4922 or Asgrowvariety A3244) by the methods described earlier, including the particlebombardment method of McCabe et al. (1988), Bio/Technology, 6:923-926 orAgrobacterium-mediated transformation (Martinell, U.S. Pat. No.6,384,301). The following constructs are introduced: (1) FAD2-1A intron(sense)-FAD2-2 intron (antisense); (2) FAD2-1A intron (sense)-FAD2-2intron (sense); (3) FAD2-1A intron (antisense)-FAD2-2 intron(antisense); (4) FAD2-1A intron (antisense)-FAD2-2 intron (sense); (5)FAD2-1B intron (sense)-FAD2-2 intron (antisense); (6) FAD2-1B intron(sense)-FAD2-2 intron (sense); (7) FAD2-1B intron (antisense)-FAD2-2intron (antisense); and (8) FAD2-1B intron (antisense)-FAD2-2 intron(sense). Transformed soybean plants are identified by selection on mediacontaining glyphosate. Fatty acid compositions are analyzed from seed ofsoybean lines transformed with the constructs using gas chromatography.Seed of the transformed plants exhibit high levels of oleic acid (over80%).

Additional linear DNA fragments containing expression constructs forsense and antisense expression of the FAD2-1, FAD2-2, and FAD3 intronsare stably introduced into soybean (Asgrow variety A4922) by the methodof McCabe et al. (1988), Bio/Technology, 6:923-926. Exemplary constructsinclude: (1) FAD2-1A intron (sense or antisense)-FAD2-2 intron (sense orantisense)-FAD3-1A intron 1 (sense or antisense); (2) FAD2-1A intron(sense or antisense)-FAD2-2 intron (sense or antisense)-FAD3-1A intron 4(sense or antisense); (3) FAD2-1A intron (sense or antisense)-FAD2-2intron (sense or antisense)-FAD3-1B intron 4 (sense or antisense); and(4) FAD2-1A intron (sense or antisense)-FAD2-2 intron (sense orantisense)-FAD3-1C intron 4 (sense or antisense). Transformed soybeanplants are identified by selection on media containing glyphosate. Fattyacid compositions are analyzed from seed of soybean lines transformedwith the constructs using gas chromatography. Seed of the transformedplants exhibit high levels of oleic acid (over 80%).

1. A transgenic soybean plant having a nucleic acid molecule comprisinga heterologous promoter operably linked to a polynucleotide that is atleast 95% identical to about 275 to about 350 contiguous nucleotides ofSEQ ID NO: 1, or complete complements thereof, wherein said soybeanplant produces seed with more oleic acid than a soybean plant having asimilar genetic background but lacking said nucleic acid molecule. 2.The transgenic soybean plant according to claim 1, wherein a seed ofsaid transformed soybean plant exhibits a modified fatty acidcomposition that is about 60-80% oleic acid.
 3. The transgenic soybeanplant according to claim 2, wherein said promoter is a seed specificpromoter.
 4. The transgenic soybean plant according to claim 1, whereinsaid polynucleotide is at least 98% identical to about 275 to about 350contiguous nucleotides of SEQ ID NO:1, or complete complements thereof.5. The transgenic soybean plant according to claim 1, wherein saidpromoter is a 7S promoter.
 6. The transgenic soybean plant according toclaim 1, wherein said polynucleotide is at least 99% identical to about275 to about 350 contiguous nucleotides of SEQ ID NO:1, or completecomplements thereof.
 7. The transgenic soybean plant according to claim1, wherein said polynucleotide is 100% identical to about 275 to about350 contiguous nucleotides of SEQ ID NO:1, or a complete complementthereof.
 8. The transgenic soybean plant according to claim 1, whereinsaid nucleic acid molecule is transcribed and is capable of selectivelyreducing the level of a transcript encoded by a FAD2-1 gene whileleaving the level of a transcript encoded by a FAD2-2 gene partiallyunaffected.
 9. The transgenic soybean plant according to claim 1,wherein said nucleic acid molecule is transcribed and is capable ofselectively reducing the level of a transcript encoded by a FAD2-1 genewhile leaving the level of a transcript encoded by a FAD2-2 genesubstantially unaffected.
 10. The transgenic soybean plant according toclaim 1, wherein a seed of said transformed soybean plant exhibits amodified fatty acid composition that is about 65-75% oleic acid.
 11. Atransgenic soybean plant having a nucleic acid molecule comprising aheterologous promoter operably linked to a nucleic acid sequence that isat least 95% identical to about 275 to about 350 contiguous nucleotidesof SEQ ID NO: 1, or complete complements thereof wherein a seed of saidtransformed soybean plant exhibits a modified fatty acid compositionthat is about 50-90% oleic acid.
 12. The transgenic soybean plantaccording to claim 11, wherein said nucleic acid sequence is transcribedand is capable of selectively reducing the level of a transcript encodedby a FAD2-1 gene while leaving the level of a transcript encoded by aFAD2-2 gene partially unaffected, substantially unaffected oressentially unaffected.
 13. A method of producing a soybean plant havinga seed with increased oleic acid content comprising: transforming asoybean plant with a nucleic acid molecule comprising a heterologouspromotor operably linked to a polynucleotide that is 95% identical toabout 275 to about 350 contiguous nucleotides of SEQ ID NO: 1, orcomplete complements thereof; and growing said soybean plant, whereinsaid soybean plant produces seed with increased oleic acid contentcompared to a soybean plant having a similar genetic background butlacking said nucleic acid molecule.
 14. The transgenic soybean plantaccording to claim 11, wherein said nucleic acid sequence is at least98% identical to about 275 to about 350 contiguous nucleotides of SEQ IDNO:1, or complete complements thereof.
 15. The transgenic soybean plantaccording to claim 11, wherein said nucleic acid sequence is at least99% identical to about 275 to about 350 contiguous nucleotides of SEQ IDNO:1, or complete complements thereof.
 16. The transgenic soybean plantaccording to claim 11, wherein said nucleic acid sequence is 100%identical to about 275 to about 350 contiguous nucleotides of SEQ IDNO:1.
 17. The transgenic soybean plant according to claim 1, herein aseed of said transformed soybean plant exhibits a modified fatty acidcomposition that is about 50% or greater of oleic acid.